paper chromatography lab hypothesis

Paper Chromatography of Plant Pigments

Learning Objectives

After completing the lab, the student will be able to:

• Extract pigments from plant material.
• Separate pigments by paper chromatography.
• Measure R f (retention factor) values for pigments.

Activity 2: Pre-Assessment

• The leaves of some plants change color in fall. Green foliage appears to turn to hues of yellow and brown. Does the yellow color appear because carotenoids replace the green chlorophylls? Explain your reasoning.
• Examine the molecular structures of photosynthetic pigments in Figure 10.1. Photosynthetic pigments are hydrophobic molecules located in thylakoid membranes. Will these pigments dissolve in water?

Activity 2: Paper Chromatography of Plant Pigments

Paper chromatography is an analytical method that separates compounds based on their solubility in a solvent.

The solvent is used to separate a mixture of molecules that have been applied to filter paper. The paper, made of cellulose, represents the stationary or immobile phase. The separation mixture moves up the paper by capillary action. It is called the mobile phase. The results of chromatography are recorded in a chromatogram. Here, the chromatogram is the piece of filter paper with the separated pigment that you will examine at the end of your experiment (see Figure 10.4).

We separate the compounds based on how quickly they move across the paper. Compounds that are soluble in the solvent mixture will be more concentrated in the mobile phase and move faster up the paper. Polar compounds will bind to the cellulose in the paper and trail behind the solvent front. As a result, the different compounds will separate according to their solubility in the mixture of organic solvents we use for chromatography.

This video demonstrates the principles and examples of chromatography. You will experiment with only paper chromatography in this lab; however, you will see that you are already familiar with some uses of thin layer chromatography.

Safety Precautions

• Work under a hood or in a well-ventilated space and avoid breathing solvents.
• Do not have any open flames when working with flammable solvents.
• Wear aprons and eye protection.
• Do not pour any organic solvent down the drain.
• Dispose of solvents per local regulations.
• Use forceps to handle chromatography paper that has been immersed in solvent and wash your hands after completing this activity.

For this activity, you will need the following:

• Plant material: intact leaves of spinach and Coleus (one leaf of each plant per pair of students)
• Filter or chromatography paper
• Ruler (one per group)
• Colored pencils
• Beakers (400 mL) (Mason jars are an acceptable substitute)
• Aluminum foil
• Petroleum ether: acetone: water in a 3:1:1 proportion
• If no hood or well-ventilated place is available, the mixture can be substituted with 95 percent isopropyl alcohol. Note that, if isopropyl alcohol is used, the pigment bands will smear. You may not be able to separate and identify the chlorophylls or carotene from xanthophyll.

For this activity, you will work in pairs .

Structured Inquiry

Step 1: Hypothesize/Predict: Discuss with your lab partner what color pigments will likely be present in the spinach leaves. Write your predictions in your lab notebook and draw a diagram of how you think the pigments will separate out on the chromatography paper.

Step 2: Student-led Planning: Read step 3 below. Discuss with your lab partner the setup of the experiment. Then agree upon the dimensions of the filter/chromatography paper that you will use. To allow good separation, the paper should not touch the walls of the container. The paper must fit inside the container while being long enough for maximum separation. Write all your calculations in your lab notebook.

Step 3: Follow the steps below to set up your filter paper and perform the chromatography experiment.

• Prepare the chromatogram by cutting a piece of filter paper. Transfer pigments from spinach leaves as in Activity 1. A heavy application line will yield stronger colors when the pigments separate, making it easier to read results. Allow the pigments to dry between applications. Wet extracts diffuse on the paper and yield blurry lines.
• Form a cylinder with the filter paper without overlapping the edges (to avoid edge effects). The sample should face the outside of the cylinder. Secure the top and bottom of the cylinder with staples.
• Pour enough separation mixture to provide a mobile phase while staying below the origin line on the chromatogram. The exact volume is not critical if the origin, the start line where you applied the solvent, is above the solvent. See Figure 10.4.

• Label the beaker with a piece of tape with your initials and your partner’s initials.
• Lower the paper into the container with the band from the extraction in the lower section. The paper must touch the solvent, but not reach the band of pigment you applied. Why must the band be above the solvent line? Write your answer in your notebook.
• Cover the container tightly with a piece of aluminum foil.
• Track the rising of the solvent front. Can you see a separation of colors on the paper?
• When the solvent front is within 1 cm of the upper edge of the paper, remove the cylinder from the beaker using forceps. Trace the solvent front with a pencil before it evaporates and disappears! Draw the colored bands seen on your chromatography paper in your lab notebook immediately. The colors will fade upon drying. If no colored pencils are available, record the colors of the lines.
• Let the paper dry in a well-ventilated area before making measurements because the wet paper is fragile and may break when handled. This is also a precaution to avoid breathing fumes from the chromatogram.
• Discard solvent mixture per your instructor’s directions. Do not pour down the drain.

Step 4: Critical Analysis: Open the dried cylinder by removing the staples. Measure the distance from the first pencil line to the solvent front, as shown in Figure 10.5. This is the distance traveled by the solvent front. Measure the distance from the pencil line to the middle point of each color band and the original pencil line. Record your results in your notebook in a table modeled after Table 10.1. The retention factor (R f ) is the ratio of the distance traveled by a colored band to the distance traveled by the solvent front. Calculate R f values for each pigment using the following equation:

R f=Distance traveled by colored band/Distance traveled by solvent front

Step 5: After determining the color of the band, tentatively identify each band. Did your results support your hypothesis about the color of each band? Discuss which aspects of the experiments may have yielded inconclusive results. How could you improve the experiment?

Guided Inquiry

Step 1: Hypothesize/Predict: What type of pigments are present in Coleus leaves and where are the different colors located? Can you make a hypothesis based on the coloration of the variegated leaves? Write your hypothesis down in your lab notebook. Would there be a difference if you performed chromatography on pigment composition from different colored regions of the leaves?

Step 2: Student-led Planning: Cut the chromatography/filter paper to the dimensions needed. Apply pigments from different parts of the Coleus leaves following the procedure described under Activity 1, keeping in mind that a darker line will yield stronger colors when the pigments are separated, which will make it easier to read the results. Allow the pigments to dry between applications. Wet extracts diffuse on the paper and yield blurry lines.

Step 3: When the solvent front reaches 1 cm from the top of the filter paper, stop the procedure. Draw the pigment bands you see on the filter paper in your lab notebook. Clearly indicate the color you observed for each band.

Step 4: Let the cylinder dry and measure the distance the front traveled from the origin and the distances traveled by each of the pigments. If the bands broadened during separation, take measurements to the middle of each band.

Step 5: Critical Analysis: Calculate R f for each of the bands and record them in a table in your notebook. Compare the R f you obtained with those of other groups. Are the R f values similar? What may have altered R f values?

Assessments

• Carotenoids and chlorophylls are hydrophobic molecules that dissolve in organic solvents. Where would you find these molecules in the cell? What would happen if you ran the chromatography in this lab with water as the solvent?
• All chlorophyll molecules contain a complexed magnesium ion. Your houseplant is developing yellow leaves. What may cause this, and how can you restore your plant’s health?
• Seeds that grow under dim light are said to be etiolated, which describes their pale and spindly appearance. They soon waste away after exhausting their food reserves. Can you explain this observation?

Lab Manual for Biology Part I Copyright © 2022 by LOUIS: The Louisiana Library Network is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License , except where otherwise noted.

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Leaf Chromatography Experiment – Easy Paper Chromatography

Leaf chromatography is paper chromatography using leaves. Paper chromatography is a separation technique. When applied to leaves, it separates the pigment molecules mostly according to their size. The main pigment molecule in green leaves is chlorophyll, which performs photosynthesis in the plant. Other pigments also occur, such as carotenoids and anthocyanins. When leaves change color in the fall , the amount and type of pigment molecules changes. Leaf chromatography is a fun science project that lets you see these different pigments.

Leaf Chromatography Materials

You only need a few simple materials for the leaf chromatography project:

• Rubbing alcohol (isopropyl alcohol)
• Coffee filters or thick paper towels
• Small clear jars or glasses with lids (or plastic wrap to cover the jars)
• Shallow pan
• Kitchen utensils

You can use any leaves for this project. A single plant leaf contains several pigment molecules, but for the most colors, use a variety of leaves. Or, collect several of each kind of leaf and compare them to each other. Good choices are colorful autumn leaves or chopped spinach.

Perform Paper Chromatography on Leaves

The key steps are breaking open the cells in leaves and extracting the pigment molecule and then separating the pigment using the alcohol and paper.

• Finely chop 2-3 leaves or several small leaves. If available, use a blender to break open the plant cells. The pigment molecules are in the chloroplasts of the cells, which are organelles encased within the plant cell walls. The more you break up the leave, the more pigment you’ll collect.
• Add enough alcohol to just cover the leaves.
• If you have more samples of leaves, repeat this process.
• Cover the container of leaves and alcohol and set it in a shallow pan filled with enough hot tap water to surround and heat the container. You don’t want water getting into your container of leaves.
• Replace the hot water with fresh water as it cools. Swirl the container of leaves around from time to time to aid the pigment extraction into the alcohol. The extraction is ready when the alcohol is deeply colored. The darker its color, the brighter the resulting chromatogram.
• Cut a long strip of coffee filter or sturdy paper towel for each chromatography jar. Paper with an open mesh (like a paper towel) works quickly, but paper with a denser mesh (like a coffee filter) is slower but gives a better pigment separation.
• Place a strip of paper into jar, with one end in the leaf and alcohol mixture and the other end extending upward and out of the jar.
• The alcohol moves via capillary action and evaporation, pulling the pigment molecules along with it. Ultimately, you get bands of color, each containing different pigments. After 30 to 90 minutes (or whenever you achieve pigment separation), remove the paper strips and let them dry.

How Leaf Chromatography Works

Paper chromatography separates pigments in leaf cells on the basis of three criteria:

• Molecule size

Solubility is a measure of how well a pigment molecule dissolves in the sol vent. In this project, the solvent is alcohol . Crushing the leaves breaks open cells so pigments interact with alcohol. Only molecules that are soluble in alcohol migrate with it up the paper.

Assuming a pigment is soluble, the biggest factor in how far it travels up the paper is particle size. Smaller molecules travel further up the paper than larger molecules. Small molecules fit between fibers in the paper more easily than big ones. So, they take a more direct path through the paper and get further in less time. Large molecules slowly work their way through the paper. In the beginning, not much space separates large and small molecules. The paper needs to be long enough that the different-sized molecules have enough time to separate enough to tell them apart.

Paper consists of cellulose, a polysaccharide found in wood, cotton, and other plants. Cellulose is a polar molecule . Polar molecules stick to cellulose and don’t travel very far in paper chromatography. Nonpolar molecules aren’t attracted to cellulose, so they travel further.

Of course, none of this matters if the solvent doesn’t move through the paper. Alcohol moves through paper via capillary action . The adhesive force between the liquid and the paper is greater than the cohesive force of the solvent molecules. So, the alcohol moves, carrying more alcohol and the pigment molecules along with it.

Interpreting the Chromatogram

• The smallest pigment molecules are the ones that traveled the greatest distance. The largest molecules are the ones that traveled the least distance.
• If you compare chromatograms from different jars, you can identify common pigments in their leaves. All things being equal, the lines made by the pigments should be the same distance from the origin as each other. But, usually conditions are not exactly the same, so you compare colors of lines and whether they traveled a short or long distance.
• Try identifying the pigments responsible for the colors.

There are three broad classes of plant pigments: porphyrins, carotenoids, and flavonoids. The main porphyrins are chlorophyll molecules. There are actually multiple forms of chlorophyll, but you can recognize them because they are green. Carotenoids include carotene (yellow or orange), lycopene (orange or red), and xanthophyll (yellow). Flavonoids include flavone and flavonol (both yellow) and anthocyanin (red, purple, or even blue).

Experiment Ideas

• Collect leaves from a single tree or species of tree as they change color in the fall. Compare chromatograms from different colors of leaves. Are the same pigments always present in the leaves? Some plants produce the same pigments, just in differing amounts. Other plants start producing different pigments as the seasons change.
• Compare the pigments in leaves of different kinds of trees.
• Separate leaves according to color and perform leaf chromatography on the different sets. See if you can tell the color of leaves just by looking at the relative amount of different pigments.
• The solvent you use affects the pigments you see. Repeat the experiment using acetone (nail polish remover) instead of alcohol.
• Block, Richard J.; Durrum, Emmett L.; Zweig, Gunter (1955).  A Manual of Paper Chromatography and Paper Electrophoresis . Elsevier. ISBN 978-1-4832-7680-9.
• Ettre, L.S.; Zlatkis, A. (eds.) (2011). 75 Years of Chromatography: A Historical Dialogue . Elsevier. ISBN 978-0-08-085817-3.
• Gross, J. (1991). Pigments in Vegetables: Chlorophylls and Carotenoids . Van Nostrand Reinhold. ISBN 978-0442006570.
• Haslam, Edwin (2007). “Vegetable tannins – Lessons of a phytochemical lifetime.”  Phytochemistry . 68 (22–24): 2713–21. doi: 10.1016/j.phytochem.2007.09.009
• McMurry, J. (2011). Organic chemistry With Biological Applications (2nd ed.). Belmont, CA: Brooks/Cole. ISBN 9780495391470.

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Leaf chromatography

In association with Nuffield Foundation

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Try this class practical using paper chromatography to separate and investigate the pigments in a leaf

Most leaves are green due to chlorophyll. This substance is important in photosynthesis (the process by which plants make their food). In this experiment, students investigate the different pigments present in a leaf, from chlorophyll to carotenes, using paper chromatography.

The experiment takes about 30 minutes and can be carried out in groups of two or three students.

• Eye protection
• Pestle and mortar
• Chromatography paper
• Beaker, 100 cm 3
• Small capillary tube (see note 1)
• Cut-up leaves, or leaves and scissors (see note 2)
• Propanone (HIGHLY FLAMMABLE, IRRITANT), supplied in a small bottle fitted with a teat pipette (see note 3)

Equipment notes

• The capillary tubing can be ‘home-made’ from lengths of ordinary glass tubing (diameter: 3–4 mm) using a Bunsen burner fitted with a flame-spreading (‘fish-tail’) jet.
• A variety of leaves can be used. Best results are obtained from trees or bushes with dark green leaves, eg holly.
• Preferably use teat pipettes that do not allow squirting, eg those fitted to dropper bottles of universal indicator.

Health, safety and technical notes

• Read our standard health and safety guidance.
• Wear eye protection throughout.
• Propanone, CH 3 COCH 3 (l), (HIGHLY FLAMMABLE, IRRITANT) – see CLEAPSS Hazcard HC085A .  The vapour of propanone is HIGHLY FLAMMABLE. Do not have any source of ignition nearby.
• Finely cut up some leaves and fill a mortar to about 2 cm depth.
• Add a pinch of sand and about six drops of propanone from the teat pipette.
• Grind the mixture with a pestle for at least three minutes.
• On a strip of chromatography paper, draw a pencil line 3 cm from the bottom.
• Use a fine glass tube to put liquid from the leaf extract onto the centre of the line. Keep the spot as small as possible.
• Allow the spot to dry, then add another spot on top. Add five more drops of solution, letting each one dry before putting on the next. The idea is to build up a very concentrated small spot on the paper.
• Attach the paper to the pencil using sellotape so that when placed in the beaker, the paper is just clear of its base.
• Place no more than about 10 cm 3 of propanone in the beaker and hang the paper so it dips in the propanone. Ensure the propanone level is below the spot.

Source: Royal Society of Chemistry

The equipment required for using paper chromatography to separate the different pigments in leaves

• Avoid moving the beaker in any way once the chromatography has started.
• Leave the experiment until the propanone has soaked near to the top, and then remove the paper from the beaker.
• Mark how high the propanone gets on the paper with a pencil and let the chromatogram dry.

Teaching notes

This experiment works very well providing care is taken over preparing the spot on the chromatography paper. It should be as small and as concentrated as possible. Encourage students to be patient and to wait until each application is dry before adding the next.

At least three spots should be obtained, and one of these should be yellow due to carotenes.

The extent to which any particular component moves up the paper is dependent not only on its solubility in propanone but also on its attraction for the cellulose in the chromatography paper. The yellow carotene spot (with a higher RF value) tends to move up the paper the furthest.

Additional information

This is a resource from the  Practical Chemistry project , developed by the Nuffield Foundation and the Royal Society of Chemistry.

Practical Chemistry activities accompany  Practical Physics  and  Practical Biology .

© Nuffield Foundation and the Royal Society of Chemistry

• 11-14 years
• 14-16 years
• Practical experiments
• Chromatography

Specification

• 2. Develop and use models to describe the nature of matter; demonstrate how they provide a simple way to to account for the conservation of mass, changes of state, physical change, chemical change, mixtures, and their separation.
• Chromatography as a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a selectively absorbent stationary phase.
• 6 Investigate how paper chromatography can be used to separate and tell the difference between coloured substances. Students should calculate Rf values.
• Chromatography involves a stationary phase and a mobile phase. Separation depends on the distribution of substances between the phases.
• The ratio of the distance moved by a compound (centre of spot from origin) to the distance moved by the solvent can be expressed as its Rf value: Rf = (distance moved by substance / distance moved by solvent)
• Mixtures can be separated by physical processes such as filtration, crystallisation, simple distillation, fractional distillation and chromatography. These physical processes do not involve chemical reactions and no new substances are made.
• Recall that chromatography involves a stationary and a mobile phase and that separation depends on the distribution between the phases.
• Interpret chromatograms, including measuring Rf values.
• Suggest chromatographic methods for distinguishing pure from impure substances.
• 12 Investigate how paper chromatography can be used to separate and tell the difference between coloured substances. Students should calculate Rf values.
• 2.11 Investigate the composition of inks using simple distillation and paper chromatography
• 2.9 Describe paper chromatography as the separation of mixtures of soluble substances by running a solvent (mobile phase) through the mixture on the paper (the paper contains the stationary phase), which causes the substances to move at different rates…
• C2.1g describe the techniques of paper and thin layer chromatography
• 2.9 Describe paper chromatography as the separation of mixtures of soluble substances by running a solvent (mobile phase) through the mixture on the paper (the paper contains the stationary phase), which causes the substances to move at different rates o…
• C5.1.4 recall that chromatography involves a stationary and a mobile phase and that separation depends on the distribution between the phases
• 3 Using chromatography to identify mixtures of dyes in a sample of an unknown composition
• C3 Using chromatography to identify mixtures of dyes in a sample of an unknown composition
• 1.9.5 investigate practically how mixtures can be separated using filtration, crystallisation, paper chromatography, simple distillation or fractional distillation (including using fractional distillation in the laboratory to separate miscible liquids…
• 1.9.7 interpret a paper chromatogram including calculating Rf values;
• carry out paper and thin-layer chromatography and measure the Rf values of the components and interpret the chromatograms;

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BIOLOGY JUNCTION

Test And Quizzes for Biology, Pre-AP, Or AP Biology For Teachers And Students

Paper Chromatography Report

Introduction The purpose of this experiment is to observe how chromatography can be used to separate mixtures of chemical substances. Chromatography serves mainly as a tool for the examination and separation of mixtures of chemical substances. Chromatography is using a flow of solvent or gas to cause the components of a mixture to migrate differently from a narrow starting point in a specific medium, in the case of this experiment, filter paper. It is used for the purification and isolation of various substances. A chromatographically pure substance is the result of the separation. Because purification of substances is required to determine their properties, chromatography is an indispensable tool in the sciences concerned with chemical substances and their reactions.

Chromatography is also used to compare and describe chemical substances. The chromatographic sequence of sorbed substances is related to their atomic and molecular structures. A change in a chemical substance produced by a chemical or biological reaction often alters the solubility and migration rate. With this knowledge, alterations or changes can be detected in the substance.

In all chromatographic separations, there is an important relationship between the solvent, the chromatography paper, and the mixture. For a particular mixture, the solvent and the paper must be chosen so the solubility is reversible and be selective for the components of the mixture. The main requirement, though, of the solvent is to dissolve the mixture needing to be separated. The porous paper used  must also absorb the components of the mixtures selectively and reversibly. For the separation of a mixture, the substances making up the mixture must be evenly dispersed in a solution, a vapor, or a gas. Once all of the above criteria have been met, chromatography can be a simple tool for separating and comparing chemical mixtures.

Hypothesis Paper can be used to separate mixed chemicals.

Materials The materials used for this lab are paper, pencil, eraser, filter paper, test tube, rubber stopper, paper clip, metric ruler, black felt-tip pen, and a computer.

Methods The first step of the method is to bend a paper clip so that it is straight with a hook at one end. Push the straight end of the paper clip into the bottom of the rubber stopper. Next, you hang a thin strip of filter paper on the hooked end of the paper clip. Insert the paper strip into the test tube. The paper should not touch the sides of the test tube and should almost touch the bottom of the test tube. Now you will remove the paper strip from the test tube. Draw a solid 5-mm-wide band about 25 mm from the bottom of the paper, using the black felt-tip pen. Use a pencil to draw a line across the paper strip 10 cm above the black band.

Pour about 2 mL of water into the test tube. The water will act as a solvent. Put the filter paper back into the test tube with the bottom of the paper in the water and the black band above the water. Observe what happens as the liquid travels up the paper. Record the changes you see. When the solvent has reached the pencil line, remove the paper from the test tube. Measure how far the solvent traveled before the strip dries. Finally, let the strip dry on the desk. With the metric ruler, measure the distance from the starting point to the top edge of each color. Record this data in a data table. Calculate a ratio for each color by dividing the distance the color traveled by the distance the solvent traveled.

Results The results of the experiment are shown in a chart and a graph.

1. How many colors separated from the black ink? Five colors separated from the black ink: yellow, pink, red, purple, and blue.

2. What served as the solvent for the ink? Water served as the solvent for the ink. As the solvent traveled up the paper, which color of ink appeared first? The color orange first appeared as the solvent traveled up the paper.

3. List the colors in order, from top to bottom, which separated from the black ink. The colors separated in this order, from top to bottom: blue, purple, red, pink, and then yellow.

4. In millimeters, how far did the solvent travel? The solvent traveled 111 mm.

5. From your results, what can you conclude is true about black ink? Black ink is a mixture of several different colors.

6 . Why did the inks separate? The inks separated because the black ink was a mixture of different pigments with different molecular characteristics. These differences allow for different rates of absorption by the filter paper.

7. Why did some inks move a greater distance? The ink least readily absorbed by the paper would then travel the farthest from the starting mark. You can conclude from this information that the different pigments were absorbed at different rates.

Error Analysis Possible errors could include inaccurate measurements of the distances traveled by the inks and mistakes when calculating the ratio traveled by the water and colors. If a longer test tube was used, a longer strip of filter paper could have been used. This may have changed the ratios. Another color may have been present, but not detected because of the filter paper length.

Conclusion The proposed hypothesis was correct. The paper chromatography did show that black ink could be separated into various colors. The black ink gets its color from a mixture of various colored inks blended together. The first color of ink to appear on the filter paper was yellow followed by pink, red, purple then blue. The colors separated the way they did because of the differences in their molecular characteristics, specifically, their solubility in water and their rate of absorption by the paper. The most soluble and readily absorbed ink color was the yellow. The least soluble and least absorbable ink color was the blue.

Where would you like to go now?

To the chromatography menu . . .

To the analysis menu . . .

To Main Menu . . .

© Jim Clark 2007 (modified July 2016)

Jove Lab Bio

Lab 7: photosynthesis — procedure.

• NOTE: In this experiment you will separate pigments from spinach leaves using chromatography paper. Individual pigments travel along the paper at different rates and may have different colors. By calculating the relative distance the pigments travel, their resolution factor, and comparing them with literature values, you can identify different pigments. HYPOTHESES: In this exercise the experimental hypothesis is that there will be multiple pigments within the spinach leaves that absorb different wavelengths of sunlight. The null hypothesis is that there is only one type of pigment within the spinach leaf.
• Use a pencil to make a line two centimeters from one end of the chromatography paper.
• Then, lay a pipe cleaner horizontally across the top of a clean 400 mL beaker.
• Place the pencil-marked side of the chromatography strip at the bottom of the beaker.
• Next, wrap the paper around the pipe cleaner so that the bottom edge is barely touching the bottom of the beaker and then secure it with a paperclip.
• When the paper is secured around the pipe cleaner, remove it from the beaker, and then place a patted-dry spinach leaf over the marked line on the chromatography paper.
• Roll a coin over the spinach leaf along the pencil line going back and forth multiple times and applying steady pressure. When the leaf is removed, a green line should be clearly present.
• Next, place 8 mL of chromatography solvent in the beaker.
• Lower the chromatography strip into the beaker so that the edge of the paper touches the solvent but the green line does not. Adjust the pipe cleaner if needed.
• Without disturbing the beaker, observe the solvent as it moves up the paper and the individual pigments separate.
• When the solvent has traveled half way up the chromatography paper, which will take approximately 10 minutes, and the pigments have separated into well-defined bands, remove the paper from the beaker.
• Mark how far the solvent traveled with a pencil and then allow the paper to dry. NOTE: The solvent evaporates quickly.
• Next, record the number of visible bands and describe their color and relative size.
• Measure how far the solvent and pigments traveled, and record this information for each pigment in Table 1. Click Here to download Table 1
• Dispose of the chromatography solvent in a waste container under a fume hood. Throw the chromatography strips into the regular trash, and then clean the beakers with soap and water.
• NOTE: In this experiment you will indirectly observe photosynthesis and cellular respiration using a floating leaf disc in a solution. During photosynthesis, air bubbles will cause the leaves to float, and during respiration, the discs will sink. HYPOTHESES: In this exercise, the experimental hypothesis is that the leaf discs will have a greater rate of photosynthesis in the bicarbonate solution, because bicarbonate provides added CO 2 to fuel photosynthesis, causing more leaf discs to float. Additionally, all of the discs will sink in dark conditions as they perform cellular respiration. The null hypothesis is that there will be no difference in the rate of photosynthesis, and therefore the number of floating discs, between the bicarbonate and water, or light and dark treatments.
• To place leaf discs under vacuum, first remove the plungers from two 20 mL syringes, and then place 10 leaf discs inside each syringe tube. Label one syringe “bicarbonate”, and label the other syringe “water”.
• Replace the plungers and push the plunger until only a small amount of air remains in the syringe. Take care not to damage the leaf discs.
• Pull 5 mL of the bicarbonate solution into one of the syringes. Invert and swirl the syringe to suspend the leaf discs in solution.
• Push as much air out as possible without expelling the solution or damaging the leaf discs.
• Then pull 5 mL of the water solution into the other syringe and swirl it as previously described (step 3).
• To create a vacuum, hold one finger over the tip of the syringe while pulling back on the plunger. Hold this for 10 seconds while swirling the syringe to keep the leaf discs in suspension.
• Then, release the vacuum. NOTE: The discs should have absorbed the solution into the air spaces in their tissues and you should see them sink. If the discs don't sink, you can repeat the vacuum creation up to three times.
• Next, add 50 mL of bicarbonate solution to a plastic cup or a glass beaker, and then gently add the discs from the bicarbonate vacuum syringe.
• For the control, add the same amount of water to an identical cup, and then add the leaf discs from the water vacuum syringe. Label the containers appropriately.
• Place both cups under a light source.
• Every five minutes record the number of discs floating on the surface of the cup in Table 3 until 20 minutes have passed. Click Here to download Table 3
• Next, remove the cups from the light source and then swirl them so that the discs at the surface intermix with any gases also at the surface.
• Move the cups to a dark place. Every five minutes record the number of leaf discs floating at the surface until 20 minutes have passed. Swirl the cup each time before placing it back in the dark.
• To clean up, dispose of the leaf discs in the trash, and pour the bicarbonate solution down the drain. Wash the syringes and cups thoroughly.
• NOTE: In the first experiment, you observed how far pigments from spinach leaves traveled on chromatography paper. Different pigments absorb light at different wavelengths.
• Using colored pens or pencils, draw the positions of the pigment bands and the solvent on Figure 3.
• Calculate the retention factor, or Rf values for the pigments, which is done by dividing the distance the pigment in question moved up the paper from the line by the distance the solvent moved up the paper from the line.
• Compare your calculated Rf values to those in Table 2 to determine the identity of the pigment. Click Here to download Table 2
• Record these data in Table 1. NOTE: In the second experiment, you observed floating and sinking leaf discs as an indirect measurement of photosynthesis and respiration.
• Graph the results with time and minutes on the x-axis and number of floating discs on the y-axis. Use two different lines to represent the water control and the bicarbonate treatment.
• Add a line to the graph to indicate the point where the discs were removed from the light condition and placed into the dark.
• Next, starting with the bicarbonate condition, use the graph to determine the point at which 50% of the leaf discs were floating. This is referred to as the effective time, or ET50. NOTE: You will notice that the discs likely hit the 50% floating mark once in the light condition and then again in the dark condition.
• Your water samples may or may not have reached the ET50 mark. If they did, add the line for this sample also.
• Finally, compare your ET50 values and graphs with the rest of the class.

Separation of Plant Pigments by Paper Chromatography

The separation of plant pigments by paper chromatography is an analysis of pigment molecules of the given plant. Chromatography refers to colour writing . This method separates molecules based on size, density and absorption capacity.

Chromatography depends upon absorption and capillarity . The absorbent paper holds the substance by absorption. Capillarity pulls the substance up the absorbent medium at different rates.

Separated pigments show up as coloured streaks . In paper chromatography, the coloured bands separate on the absorbent paper. Chlorophylls, anthocyanins, carotenoids, and betalains are the four plant pigments.

This post discusses the steps of separating plant pigments through paper chromatography. Also, you will get to know the observation table and the calculation of the Rf value.

Content: Separation of Plant Pigments by Paper Chromatography

Paper chromatography, plant pigments, steps of plant pigment separation, observation, calculation.

It is the simplest chromatography method given by Christian Friedrich Schonbein in 1865. Paper chromatography uses filter paper with uniform porosity and high resolution.

The mixtures in compounds have different solubilities . For this reason, they get separated distinctly between the stationary and running phase.

• The mobile phase is a combination of non-polar organic solvents. The solvent runs up the stationary phase via capillary movement.
• The stationary phase is polar inorganic solvent, i.e. water. Here, the absorbent paper supports the stationary phase, i.e. water.

Plant pigments are coloured organic substances derived from plants. Pigments absorb visible radiation between 380 nm (violet) and 760 nm (red).

They give colour to stems, leaves, flowers, and fruits. Also, they regulate processes like photosynthesis, growth, and development.

Plants produce various forms of pigments. Based on origin, function and water solubility, plant pigments are grouped into:

• Chlorophylls (green)
• Carotenoids (yellow, orange-red)
• Anthocyanins (red to blue, depending on pH)
• Betalains (red or yellow)

Chlorophyll : It is a green photosynthetic pigment. Chlorophyll a and b are present within the chloroplasts of plants. Because of the phytol side chain, they are water-repelling . Their structure resembles haemoglobin. But, they contain magnesium as a central metal instead of iron.

Carotenoids : These are yellow to yellow-orange coloured pigments. Also, they are very long water-repelling pigments. Carotenoids are present within the plastids or chromoplasts of plants.

Anthocyanins : These appear as red coloured pigments in vacuoles of plant cells. Anthocyanins are water-soluble pigments. They give pink-red colour to the petals, fruits and leaves.

Betalains : These are tyrosine derived water-soluble pigments in plants. Betacyanins (red-violet) and betaxanthins (yellow-orange) are the two pigments coming in this category. They are present in vacuoles of plant cells.

You can separate all the above pigments using paper chromatography.

Video: Separation of Plant Pigments

Preparation of Concentrated Leaf Extract

• Wash spinach leaves in distilled water.
• Then take out the spinach leaves and allow the moisture to dry out.
• After that, take a scissor and cut the leaves into the mortar.
• Take a little volume of acetone into the mortar. Note : Acetone is used instead of water to mash the leaves because it is less polar than the water. This allows a high resolution of the molecules in the sample between the absorbent paper.
• Then, grind spinach leaves using a pestle until liquid paste forms. Note : The liquid in the crushed leaf paste is the pigment extract.
• After that, take out the mixture into the watch glass or Petri dish.

Load the Leaf Extract onto Absorbent Paper

• Take Whatman filter paper and draw a line above 2 cm from the bottom margin. You can use a pencil and scale to draw a fainted line. Note : A pencil is used because pencil marks are insoluble in the solvent.
• Then, cut the filter paper to make a conical edge from the line drawn towards the margin end. You can use a scissor to cut the Whatman filter paper. Note : The conical end at the bottom of the filter paper results in better separation.
• Put a drop of leaf extract on the centre of a line drawn on the absorbent paper.
• Then, at the same time dry the absorbent paper.
• Repeat the above two steps many times so that the spot becomes concentrated enough.

Setup the Chromatography Chamber

• Take a clean measuring cylinder and add rising solvent (ether acetone) up to 4 ml.
• Bend the strip of paper from the top. Then, using a pushpin attach the paper to the bottom of the cork.
• Adjust the length of the paper. The absorbent paper should not touch the surface of the measuring cylinder.
• After that, allow the solvent to move up the absorbent paper.
• When the solvent front has stopped moving, remove the paper.
• Allow it to dry for a while until the colours completely elute from the paper.
• At last, mark the front edge travelled by each pigment.

Over the dried paper strip, you will see four different bands. Different colour streaks form because of different affinities with the mobile phase (solvent).

• The carotene pigment appears at the top as a yellow-orange band.
• A yellowish band appears below the carotene, which indicates xanthophyll pigment.
• Then a dark green band represents the chlorophyll-a pigment.
• The chlorophyll-b pigment appears at the bottom as a light green band.

Observation Table

1. Light green spot indicates chlorophyll-b pigment.

• Rf value= Distance chlorophyll-b travelled / Distance solvent travelled = 2/10 = 0.2

2. Dark green spot represents chlorophyll-a pigment.

• Rf value= Distance chlorophyll-a travelled / Distance solvent travelled = 3.7/10 = 0.37

3. The yellow band represents xanthophyll pigment.

• Rf value= Distance xanthophyll travelled / Distance solvent travelled = 5.6/10 = 0.56

4. The yellow-orange band indicates carotene pigment.

• Rf value= Distance carotene travelled / Distance solvent travelled = 9/10 = 0.9

Factors affecting the Rf values of a particular analyte are:

• Stationary phase
• The concentration of the stationary phase
• Mobile phase
• The concentration of the mobile phase
• Temperature

The Rf value of compounds in the mixture differs by any changes in the concentration of stationary and mobile phases.

Temperature affects the solvent capillary movement and the analyte’s solubility in the solvent. Rf value is independent of the sample concentration. Its value is always positive .

Related Topics:

• Difference Between Budding and Grafting
• Phototropism in Plants
• Potometer Experiment

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Nice experiment and understanding.

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2: Paper Chromatography of Gel Ink Pens (Experiment)

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• Page ID 93983

• Santa Monica College

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• To use paper chromatography to identify whether certain colored inks are pure substances or mixtures.
• To obtain a paper chromatogram of various gel inks
• To identify components of inks by calculating R f values

Chromatography is a method of physically separating mixtures into its individual components. It is a common laboratory technique used to identify unknown components in mixtures.

There are several types of chromatography; all types employ a mobile phase or eluent (it can be liquid or gas), which is forced through a stationary phase (a solid or semi-solid). Mixtures are separated because some components will be more attracted to the stationary phase (and stick to it) while some components will be more attracted to the mobile phase (and travel with it).

By eye, we cannot know if each ink color is a mixture or pure substance. Using chromatography, the components in a sample will migrate along the filter paper at different rates such that they become spread out and separated from each other. The mobile phase takes advantage of differing solubility or polarity of the components in order to separate them. This component traveling process is called elution . Chromatography can be used to separate inks, dyes, pigments in plants, or used to determine the chemical composition of many substances.

Figure 1 shows a beaker containing mobile phase and a prepared paper stationary phase. A line drawn at the bottom edge of the paper is the starting line . The starting line and ink dots must be above the level of the mobile phase when the paper is placed inside the beaker. If the starting line is below the liquid level, the inks will wash out into the mobile phase rather than elute up the stationary phase. Another line is drawn about 10 cm above the bottom edge of the paper. This is the finish line . Its location was chosen for this experiment because when the eluting solution reaches that line, any inks that are mixtures should be clearly separated.

When the solvent front reaches the finish line, the paper should be removed immediately from contact with the mobile phase.

Figure 2 shows a typical paper chromatogram. There are a few difficulties commonly encountered in the elution process. One problem is that spots tend the spread out as they elute, and can bleed into each other as they proceed up the paper. This can be confusing when interpreting the chromatogram. To avoid this problem, space the spots of sample far apart and make repeated, tiny applications of sample to prevent spreading. Another problem is an uneven solvent front. This can happen if the beaker is nudged – if the mobile phase sloshes inside, the elution trails may travel diagonally, which makes interpretation very difficult. This can also happen if the two edges of the chromatogram are allowed to touch when they are stapled or taped together to form a cylinder.

A component with a given solubility travels along with the mobile phase at one rate, regardless of what other components are present in the sample. If the red part of purple ink travels at the same rate as pure red ink, and both stop in the same place, the two should be the same red ink. The two red spots should have the same Retention Factor , R f . The R f is the distance, \(D\), traveled by the spot divided by the distance traveled by the eluting solution, or Solvent Front , \(F\).

\[R_f=\frac{D}{F} \label{1}\]

Comparing the R f values allows the confirmation of a component in multiple samples because unique components have unique R f values.

Materials and Equipment

Materials: chromatography paper, gel pens, and eluting solution.

Equipment: 600-mL beaker, pencil, ruler, plastic wrap, tape and paper towels.

Wear safety goggles at all times. Use eluting solution only in the hood. Do not breathe fumes from the eluting solution. Be sure to handle only the dry part or the chromatogram when removing it from the beaker. Wash hands thoroughly if the eluting solution touches your skin. Personal protective equipment (PPE) required: lab coat, safety goggles, closed-toe shoes, gloves

Part A: Preparation of Chromatography Paper

• Wash your hands thoroughly to remove excess oils from your skin. Obtain a ruler and a piece of chromatography paper from your instructor. Handle the paper only on the edges to avoid leaving fingerprints, as these may hinder the elution process.
• Place the chromatography paper on a sheet of clean notebook paper or paper towel to avoid picking up dirt or contaminants from the bench top. Orient the paper into a “landscape” position and write your name on the top edge of the paper in one corner. Using a pencil and ruler to measure accurately, draw a straight line across the paper, about 1.5 cm above the bottom edge. This is the starting line . Draw another line about 10 cm above the bottom edge. This is the finish line .
• On the starting line, measure in from one side about 2.5 cm and lightly draw a small “X” centered on the starting line. Draw seven more, 1.5 cm apart.
• In the center of each X, make a small spot of ink color in this order:

black, burgundy, red, pink, violet, turquoise, green, blue

When you have finished, you should have something that looks like Figure 3.

Figure 3: Prepared Chromatography Paper

• Go back over each ink spot a second time to ensure there is enough ink in the spot.
• Obtain a small piece of tape and gently curl the paper into a cylinder, with the spots on the outside. Tape the ends together near the top and bottom, taking care that the two edges of the paper do not touch. If they do touch, the eluent will creep on a diagonal, and the spots will run together, or not in straight lines.

Part B: Acquisition of Chromatogram

• Take a 500-mL beaker and pour about 25-mL of eluting solution into the beaker. Obtain a piece of plastic wrap to cover the top.
• Gently place the paper cylinder into the beaker and cover the top with the plastic wrap. Remember that the spots must be above the liquid level for the experiment to work. Watch the eluent creep up the paper until it begins to move some of the ink. It will take about 45-90 minutes for the solvent front to reach the finish line.
• When the solvent front reaches the finish line, remove the paper from the beaker, being careful to touch only the top. Let excess eluent drip into the beaker. Gently remove the tape and lay the chromatogram on a piece of paper towel in the hood. Leave the paper in the fume hood, where it will dry completely. If needed, use a heat lamp (in the fume hood) to dry the chromatogram; if using the heat lamp, allow 5-10 minutes to dry.

Part C: Interpretation of Chromatogram

• Write the names of the original ink colors beneath each “X” mark (from the order you added the colors). Each ink sample should no longer be on the “X” mark, and have travelled up the paper, becoming one or more separate color spots between the starting and finish lines. Circle around each color spot.
• Use a ruler and draw a plus sign in the center of each spot. Measure the distance from the starting line to each plus sign. Record this distance for each spot on your lab report. These are the \(D\) values, in cm.
• Measure the distance between the starting line and the finish line or, the farthest up that the solvent front reached. Record this distance. This is the \(F\) value, in cm.
• Calculate the retention factor (R f ) for each spot and record the values in your lab report.
• You and your lab partner will hand in your lab reports at the same time, with the paper chromatogram stapled to the lab reports.

Lab Report: Paper Chromatography of Gel Ink Pens

Experimental data.

\(F\) value (the distance traveled by the eluting solution):

Record \(D\) values for each eluted spot to the nearest 0.1 cm. Draw an X through any unused boxes.

Calculate and record the R f value for each eluted spot, using Equation \ref{1}:

Show calculations for R f below for the Purple marker:

Data Analysis

• This lab employs chromatography to separate the components in ink. What other applications can we use chromatography for?
• Are any of the R f values in the table above the same (or similar, within 0.1)? What would the same or similar R f values indicate?
• What are the units for R f values?
• Record ink colors in the appropriate column; for mixtures, list the colors of the components.
• Which color travelled farthest with the mobile phase? What does this indicate about that component’s attraction to the mobile phase? To the stationary phase?
• Which of the materials tested (gel ink pen and colored markers) had an R f value of zero? What does this indicate about that component’s attraction to the mobile phase? To the stationary phase?
• Why is it necessary to use a pencil to mark the lines and X’s on the paper?
• The air we breathe is a mixture of different components. What is the composition of dry air, by percentage? See Fig 1.2 in Chapter 1 of the text.

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CRISPR–Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing

• Published: 15 August 2022
• Volume 87 , pages 777–788, ( 2022 )

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• Irina Gostimskaya 1  

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The development of a method for genome editing based on CRISPR–Cas9 technology was awarded The Nobel Prize in Chemistry in 2020, less than a decade after the discovery of all principal molecular components of the system. For the first time in history a Nobel prize was awarded to two women, Emmanuelle Charpentier and Jennifer Doudna, who made key discoveries in the field of DNA manipulation with the CRISPR–Cas9 system, so-called “genetic scissors”. It is difficult to overestimate the importance of the technique as it enables one not only to manipulate genomes of model organisms in scientific experiments, and modify characteristics of important crops and animals, but also has the potential of introducing revolutionary changes in medicine, especially in treatment of genetic diseases. The original biological function of CRISPR–Cas9 system is the protection of prokaryotes from mobile genetic elements, in particular viruses. Currently, CRISPR–Cas9 and related technologies have been successfully used to cure life-threatening diseases, make coronavirus detection tests, and even to modify human embryo cells with the consequent birth of babies carrying the introduced modifications. This intervention with human germplasm cells resulted in wide disapproval in the scientific community due to ethical concerns, and calls for a moratorium on inheritable genomic manipulations. This review focuses on the history of the discovery of the CRISPR–Cas9 system with some aspects of its current applications, including ethical concerns about its use in humans.

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A HISTORY OF THE DISCOVERY OF THE MAIN COMPONENTS OF THE CRISPR–Cas9 SYSTEM

CRISPR – clustered regularly interspaced short palindromic repeats – were first discovered in the sequences of DNA from Escherichia coli bacteria and described in 1987 by Ishino et al. [ 1 ] from Osaka University (Japan). At that time sequencing of these difficult-to-study DNA fragments took several months, but neither their origin nor their significance in the bacterial cell were understood by their discoverers. Although in the early work in this field, the biological function of the CRISPR system had not yet been elucidated, scientists had already proposed a way to use the information encoded in CRISPR loci in medical research, namely, for genotyping various strains of bacteria: initially on Mycobacterium tuberculosis [ 2 ], and later on Streptococcus pyogenes [ 3 ]. As it turned out, CRISPR loci had a high degree of polymorphism in different strains of the same species of pathogenic bacteria, which enabled the identification of bacterial strains in clinical conditions.

A significant breakthrough in understanding the biological function of CRISPR loci occurred with the discovery of Francisco Mojica of the University of Alicante (Spain), who came across similar structures in the archaeal genome of Haloferax mediterranei in 1995 [ 4 ]. Their presence in two evolutionarily remote domains of life suggested these elements’ great functional significance, and served as an impetus for further research. Mojica noticed the similarity of the elements he described in archaea with previously found DNA repeats in bacterial genomes, and was one of the first scientists to hypothesize that these unusual loci include fragments of foreign DNA and are, in fact, a part of the immune system of bacteria and archaea [ 5 ]. In the same year as Mojica, two other laboratories independently reached similar conclusions [ 6 , 7 ], announcing the beginning of an era of active research into this extraordinary natural phenomenon. In line with the theory of the prokaryotic immune system, viral DNA fragments (“spacers” 17-84 bases long), separated by short palindromic repeats (23-50 bases [ 8 ]) and grouped into clusters in intergenic regions, represent a library of potentially dangerous genetic information (for an overview of the microbial antiviral arsenal, see reviews by Isaev et al. [ 9 , 10 ]). Initially, it was assumed that such a system would work by the mechanism of RNA interference. However, in the publication of Marraffini and Sontheimer, it was experimentally demonstrated for the first time that the actual target of the immune system of prokaryotes was foreign DNA [ 11 ], and not mRNA, and, therefore, the use of such a system in the laboratory could represent a potential tool for genomic editing. Interestingly, later studies demonstrated that some of the described CRISPR systems do work with RNA molecules directly [ 12 , 13 ] and, therefore, can be used to deactivate specific transcripts inside the cell in a selective way [ 14 , 15 ].

The first experimental information about the mechanism of action of the CRISPR system was obtained in 2007 in the studies of two French food scientists, Rodolphe Barrangou and Philippe Horvath, who worked with yoghurt cultures of bacteria Streptococcus thermophilus for the Danish company Danisco [ 16 ]. Due to the company’s rich collection of bacterial strains collected since the 1980s, scientists have been able to trace the historical course of the bacterial acquisition of spacers at the CRISPR locus in response to viral attacks by bacteriophages. The addition of new spacers in this work caused acquired immunity to the corresponding new types of bacteriophages in S. thermophilus : observation which subsequently led to the authors obtaining one of the first patents in this area [ 17 ] and the start of bacterial cultures’ “vaccination” with the use of CRISPR-based technology by Danisco in 2005 [ 18 ].

Currently, CRISPR repeats have been found in most archaeal genomes and nearly half of the studied bacterial ones, but they have not been found in eukaryotic or viral DNA sequences. The existence of CRISPR repeats in mitochondria was suggested in one of the earliest publications on the subject (the same article described CRISPR in cyanobacteria for the first time) [ 19 ]. The authors used a set of previously published data on the sequencing of mitochondrial plasmids from Vicia faba L. beans [ 20 ], and their conclusions were further cited by Mojica et al. [ 21 ], but these observations were not confirmed in later studies [ 8 ].

At the time of initial discoveries, a variety of different acronyms was used for CRISPR by individual scientific groups, which presently complicates the search for early articles on the topic. The current name for CRISPR first appeared in Jansen et al. [ 22 ] in 2002 and was suggested by Mojica in correspondence between the two collaborating scientific groups. The same publication was the first one to describe the presence of genes associated with CRISPR repeats (named by the authors cas1-4 , CRISPR-associated genes). These genes were found in close proximity to the CRISPR loci of various prokaryotes, and two of them contained motifs characteristic of helicase and nuclease, which supported the authors’ hypothesis about the non-random association of the cas genes with the CRISPR locus, and their involvement in DNA metabolism. Also in 2002, the same neighborhood of genes was described by a team of scientists led by Eugene Koonin from the NCBI Institute (Bethesda, USA), but the association of these genes with CRISPR arrays was not discerned by them at the time [ 23 ]. From the moment of the first discovery of genes associated with the CRISPR system, to the present day, their truly extraordinary abundance and diversity have been found in prokaryotic cells, including representatives of the families of helicases, nucleases, polymerases, and others. Proteins associated with this system can be assigned to either the adaptive module (participating in the acquisition of immunity, main representatives – Cas1 and Cas2), or the effector module (directly involved in the destruction of mobile genetic elements through their recognition and cleavage), with some additional and regulatory proteins also found to be associated with the system [ 24 ]. At present, a way of classification is recognized in which all currently known CRISPR–Cas systems are divided into 2 classes and 6 types, which, in turn, are also divided into numerous subtypes: at the time of writing the review, Makarova et al. [ 25 ] described >30 subtypes ( Fig. 1 ). The main difference between the classes is that the effector module of Class 1 systems is represented by a complex of several proteins, while in Class 2 it is a single multidomain protein (Cas9, Cas12, or Cas13) [ 26 - 28 ].

Conventional classification of known CRISPR–Cas systems.

Of all the known Cas proteins, the most studied ones are the proteins belonging to the system of directional cutting of foreign DNA (and, as it was found out later, in some cases, RNA), the so-called “genetic scissors”, among which is the nuclease Cas9. This protein was first described in connection with its association with CRISPR repeats in an article by Bolotin et al. [ 6 ], where it was originally named Cas5 (other alternative names are Csn1 and Csx12). In addition, the authors identified the presence of the HNH motif (His-Asn-His), which is also found in other nucleases. Another important observation made by Bolotin et al. was the discovery of a specific pattern in the nucleotide sequences on one side of the described spacers of the CRISPR arrays, but the understanding of the role for this phenomenon was only revealed in later studies. Currently, short motifs adjacent to protospacers but absent in the original spacers of the CRISPR locus are called PAMs (protospacer adjacent motifs) [ 29 ]. Protospacers are DNA fragments that are attacked by the immune system of prokaryotes, and are identical to the corresponding spacers at the CRISPR locus, except for the PAM motif. These motifs are important at the stage of recognition of potentially dangerous genetic information; their presence at the end of the sequence signals that the DNA fragment is foreign and needs to be destroyed, while the DNA sequences stored in the CRISPR locus as spacers and not containing PAM motifs are not attacked by the prokaryotic immune system.

A crucial player in the CRISPR–Cas9 system turned out to be a short RNA molecule, a processed product of transcription from the CRISPR locus that directs proteins of the prokaryotic immune system to foreign molecules with genetic information. A group of researchers led by John van der Oost (Wageningen University, the Netherlands), who described the existence of such RNA molecules, gave them the name crRNA (CRISPR-associated RNA). It was also noted that the initial result of transcription from the CRISPR locus is a pre-crRNA precursor molecule consisting of several spacers and repeats, which is later cleaved into individual fragments [ 30 ]. In the work of the group led by Virginijus Siksnys (Vilnius University, Lithuania), it was demonstrated that the length of the actual “guide” crRNA sequence of 20 base pairs, complementary to the target DNA, is necessary and sufficient for the nuclease activity of the CRISPR–Cas complex, even if the spacer in CRISPR locus is represented by a longer sequence of nucleotides [ 31 ]. This publication was one of two in vitro studies, carried out in parallel and independently in competing laboratories, that described, for the first time, how the Cas9 enzyme uses crRNA to attack foreign DNA.

The final missing piece in the puzzle, without which it is impossible to assemble a working CRISPR–Cas9 system in vitro , turned out to be another short RNA molecule, discovered in connection with its participation in crRNA processing by Emmanuelle Charpentier’s group in 2011 [ 32 ]. This molecule, essential for nuclease activity, was named tracrRNA (trans-activating CRISPR RNA). In subsequent work, ultimately acknowledged by the Nobel Prize, the role of tracrRNA in the mechanism of target DNA cutting was shown. It was also proposed at the time that two RNA molecules, crRNA and tracrRNA, could be combined into one chimeric molecule (sgRNA – single guide RNA), which greatly facilitated the practical use of the CRISPR–Cas9 system in subsequent applications [ 33 ]. Figure 2 shows the timeline of the historical events in the discovery of the CRISPR–Cas9 system’s components: initially the CRISPR locus itself, then the proteins associated with it, including Cas9, and later, two RNA molecules necessary for the formation of the ribonucleoprotein complex and recognition of substrate DNA.

Historical timeline of discoveries of the components of the CRISPR–Cas9 system. 1987 – Short DNA repeats, later called CRISPR, were first noticed in bacterial genomes, and, in 1995, also found in archaea. 2005 – The role of CRISPR loci in the protection of prokaryotes from foreign genetic information was proposed, and the Cas9 protein was described for the first time (initial information on proteins associated with the CRISPR locus appeared in 2002). Two RNA molecules, crRNA and tracrRNA, were discovered as part of the complex in 2007 and 2011, respectively. The Nobel Prize-winning work, where all of the components were assembled in vitro and two RNA molecules combined into one strand for the ease of use of the system, was published in 2012.

USE OF THE CRISPR–Cas9 SYSTEM IN EUKARYOTIC CELLS

The discovery of the necessary and sufficient components of the CRISPR–Cas9 system started a race to be the first to apply the system to the genetic editing of human and animal cells. In January 2013, almost simultaneously, five research articles authored by different research teams appeared, all reporting that they had achieved the goal. Two publications from the same issue of the journal Science , offering probably the best approach to the problem had been produced by the laboratories of George Church (Harvard University, USA) and Feng Zhang (Broad Institute, USA). In these publications, it was shown that for successful DNA editing in human cells, it was necessary to carry out several steps: these include codon optimization and the addition of a nuclear localization signal to the cas9 gene, lengthening of the sgRNA molecule (to improve the efficiency of the system), as well as the possible addition of a DNA template for homologous recombination with which the cells can repair the DNA double break (the last step was described only by the group of G. Church) [ 34 , 35 ]. Also in January 2013, similar publications came out from the laboratories of Jennifer Doudna (Berkeley College, USA) [ 36 ], Jin-Soo Kim (Seoul University, South Korea) [ 37 ] and J. Keith Joung (Harvard School of Medicine, USA) [ 38 ]. In the last article [ 38 ], the described work was carried out on zebrafish rather than human cells but, importantly, the use of the CRISPR–Cas9 system on germline cells was demonstrated for the first time.

FIRST CRYSTALLOGRAPHIC STUDIES

The most studied protein from the Cas group is the Cas9 nuclease; in the ~20 years since the discovery of the cas genes more than 20,000 articles in the PubMed system mention the name Cas9 in one context or another. Attempts to obtain detailed information about the structure of this protein resulted in the first two crystallographic studies being published almost simultaneously: in February 2014 two crystal structures of Cas9 appeared in the database PDBe (“Protein Data Bank in Europe”), and the accompanying articles were published in the journals Nature and Cell [ 39 , 40 ]. The structure that came out of the laboratory of Jennifer Doudna was of an apo-protein (PDBe ID 4cmp, PDBe DOI: https://doi.org/10.2210/pdb4cmp/pdb ), while the research group of Osamu Nureki (University of Tokyo, Japan) succeeded in crystallising the protein in a complex with a “guide”-RNA and “target”-DNA (PDBe ID 4oo8, PDBe DOI: https://doi.org/10.2210/pdb4oo8/pdb ).

These, as well as many subsequent studies, used the Cas9 protein from S. pyogenes , SpCas9, which consists of 1368 amino acids and is a multidomain and multifunctional endonuclease. Crystal structures revealed that the Cas9 protein is spatially divided into 2 lobes: a target recognition lobe and a nuclease lobe, with the guide RNA and target DNA occupying the positively charged groove at their interface. The key structures of the nuclease lobe of SpCas9 are 2 domains: HNH and RuvC, each of them cleaves one of the target DNA strands. Figure 3 shows the general architecture of the SpCas9–sgRNA–DNA complex, where the complex secondary structure of the bound RNA molecule, and the unwound state of the double-stranded DNA molecule with the formation of a DNA–RNA heteroduplex can be seen (PDB ID 5F9R, PDB DOI: https://doi.org/10.2210/pdb5F9R/pdb , [ 41 ]). At the time of writing, hundreds of crystal structures of the Cas9 family proteins are available from the PDB, PDBe, and PDBj databases.

Three-dimensional organization of the Cas9 protein in the complex with “guide” RNA (sgRNA) and substrate (Target DNA), crystallographic data (PDB ID 5F9R, PDB DOI: https://doi.org/10.2210/pdb5F9R/pdb ).

PATENT DISPUTE

The understandable motive of individual scientists, as well as organizations involved in the study of the CRISPR–Cas9 system, was the possible financial gain potentially obtainable from the use of this promising technology. One of the first patent applications was filed jointly by the University of California at Berkeley, representing Doudna, the University of Vienna (where one of the two lead authors from the key publication on CRISPR–Cas9 worked [ 33 ]), and Charpentier as an individual inventor in accordance with the rules of the University of Umeå (Sweden), where Charpentier worked at the time of publication of the article [ 18 ]. This patent application was filed in May 2012 [ 42 ], while in December 2012 Zhang and the Broad Institute also submitted a patent application [ 43 ] simultaneously with the acceptance of Zhang’s paper on human cells’ editing for publication in Science [ 35 ]. Initially, it was Zhang’s application that turned out to be successful and resulted in a patent in April 2014, while Doudna’s application was still pending at that time. Doudna’s team disagreed with the decision, after which a long dispute between the two parties followed, including appeals and court hearings which ultimately led to an ambiguous situation in CRISPR–Cas9 licensing. Due to the fact that by 2019 both competing parties had patents in this area, some of the biotech companies that used the CRISPR–Cas9 system on human cells received a license from the team of Doudna, while others – from Zhang. However, the U. S. Patent and Trademark Office Appeal Board in February 2022 again confirmed the priority of Zhang and the Broad Institute in the position of the patent holder for the use of CRISPR–Cas9 in human cells, which caused disappointment and frustration from the opposing side, and financial complications for companies licensed by the team of Doudna [ 44 ]. Doudna and Charpentier, however, won a similar dispute in Europe, and also hold major patents on the use of technology in the U.K., China, Japan, Australia, New Zealand, and Mexico [ 18 ].

GENE THERAPY AND ETHICAL ISSUES ASSOCIATED WITH IT

The haste with which competing laboratories sought to bring their research to the public’s attention, as well as the race to patent this technology, were indicators of the significance of this scientific breakthrough. Undoubtedly, one of the main driving forces that motivated many scientists to take part in research using this particular technology was the potential of modifying human cells, both somatic and germline. However, despite the apparent advantages of the CRISPR–Cas9 system, numerous ethical and technical difficulties stand in the way of researchers who dream of curing life-threatening diseases, especially if the genetic changes resulting from such manipulations can be inherited.

Gene therapy was administered for the first time in September 1990: a four-year-old girl suffering from adenosine deaminase (ADA) deficiency received an infusion of genetically engineered T-lymphocytes. Cells taken from the girl’s blood were modified using a viral vector – a deactivated virus that carries a healthy copy of the gene. As journalists who covered the story noted “rarely in modern medicine has an experiment been filled with so much hope”, and the doctor who performed this procedure, W. French Anderson, became known as the “father of gene therapy”. As time went on, however, the disturbing evidence of the adverse side effects of some attempts at gene therapy in both animals and humans began to accumulate. The tragic story of Jesse Gelsinger, an American teenager from Philadelphia who died from the effects of gene therapy in 1999, shocked the world and caused widespread skepticism and a significant delay in the development of the technology. In the case of Gelsinger, a large-scale autoimmune response of the body to a viral vector carrying the ornithine transcarbamylase gene led to a sharp increase in body temperature, renal and pulmonary failure, jaundice, impaired blood clotting, and subsequent death within only four days from the moment of gene therapy administration [ 45 ].

Extensive discussions of the safety and, importantly, the ethical issues arising from the possibility of potential gene therapy with CRISPR–Cas9 began soon after the first publications showing this system’s use in human cells. One of the first steps in initiating formal discussions was taken by Doudna, who organized a conference on scientific, medical, legal, and ethical issues related to the genomic modification, held in the Napa Valley in California in January 2015. A subsequent report of the results of the conference was published in March 2015 in the journal Science [ 46 ], which essentially carried recommendations to strongly discourage work on introducing heritable changes in human embryonic cells, at least for the duration of active discussions of the social, environmental and ethical consequences of such manipulations. Almost simultaneously with this report, a comment was also published in the journal Nature about the serious risks linked to creating heritable changes in human embryos [ 47 ]. The authors expressed concerns that premature work on embryonic cells could have a negative impact on the field of gene therapy in general, and could set back the work of researchers attempting to treat genetic and infectious diseases in somatic cells for years. The March 2015 report from the Napa conference and the commentary in Nature urging not to edit the human embryonic genome were released amidst growing agitation in the scientific community over leaked news that such experiments had actually already been carried out. A group of scientists from Sun Yat-sen University (Guangzhou, China), after unsuccessful attempts to get their manuscript accepted by the journals Nature and Science , in April 2015 finally published their article on the use of the CRISPR–Cas9 system on human embryonic cells [ 48 ]. The researchers emphasized that they used non-viable embryos obtained by the fusion of two sperm cells with one egg and, therefore, discarded by in vitro fertilization (IVF) laboratories. The main conclusion of the article was that the CRISPR–Cas9 technology at the time of the study was not yet ready for use on human embryonic cells due to the identified shortcomings in the system’s efficiency and specificity. A comment of the journal Protein & Cell (Beijing, China), that published this work, stated that the article (in addition to its scientific value) would promote an open exchange of information about current research in the area; and despite the ambiguity of the issue and conflicting opinions on the topic, the publication would stimulate the necessary discussions about genomic editing of germline cells. Interestingly, the manuscript had been sent to Protein & Cell together with the references obtained during previous attempts to publish the work, and was accepted by the editors for publication within two days from the date of submission. The subsequent debate in the scientific community was described as “epic” [ 49 ] and provoked interest in this complex issue from the wider public, as well as in governmental and regulatory organizations in various countries.

The notorious scandals caused by the conduct of medical experiments on humans in the past have led to the creation of general international guidelines on bioethics. The best-known documents in this area are the Nuremberg Code, developed after the trial of Nazi doctors in 1947, and the subsequent Declaration of Helsinki from 1964, which expanded the principles of the code and detailed the application of these principles to clinical research. Another important document, the Belmont Report, was issued by the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research in the United States in 1978. This commission was created in the wake of shocking revelations of an inhumane syphilis study from 1932 to 1972 in Tuskegee. For decades, hundreds of impoverished African-American men infected with syphilis have been studied for the progression of their disease. Although penicillin had become the standard treatment for syphilis by 1947, it was not offered to study participants, despite the obvious physical suffering of the patients and the continued spread of the infection in their families.

The Nuremberg Code, the Declaration of Helsinki and the Belmont Report are based on the basic ethical principles of biomedical research, such as respect for the individual, informed consent of the patient, understanding of the risks and benefits, voluntary participation, fairness in the conduct of experiments, maximum professionalism of the researchers, etc. These principles, and their application in medical practice, are relevant to the events of November 2018, when the Chinese scientist Jiankui He announced the birth of babies who, for the first time, had undergone gene modification using the CRISPR–Cas9 system. The injection of this system into the mother’s egg was made at the stage of the IVF procedure immediately after the fusion of the sperm, and therefore all the changes potentially introduced into the genome during this procedure would be heritable. The world scientific community was shocked at how premature such medical experiments were, and the high degree of risk taken by the researchers conducting the experiment. In particular, scientists were worried about the possibility of creating unplanned (“off-target”) mutations in the genome of future babies. At the time of the experiment He (also known under the shortened name JK – from Jiankui) was not a well-known figure in the CRISPR–Cas9 community, however, after the announcement of his experiments, he attracted world-wide attention. He studied physics at the University of Science and Technology (Hefei, China) and then moved to the United States, where he received his PhD under the supervision of Michael Deem, Professor of Physics, Astronomy and Bioengineering at Rice University (Houston, Texas), and later worked as a post-doc at Stanford University (California) in the laboratory of Professor Stephen Quake. In the group of Deem He used the methods of theoretical biophysics, mathematical modelling and computer simulations, publishing papers on, among other things, influenza virus strains and spacer sequences in CRISPR loci [ 50 , 51 ], while in the laboratory of Quake, he learned the methods of molecular biology and became interested in the innovative technologies of Silicon Valley. Returning to China, He continued his collaboration with Deem, and also successfully implemented the innovative ideas in the field of DNA sequencing of his second supervisor, Quake, creating a successful company Direct Genomics based on the technology [ 18 , 52 ]. In China, he became quite famous as a young scientist and successful entrepreneur who had returned from abroad under the Thousand Talents program. He received a position and a laboratory at the Southern University of Science and Technology (SUStech, Shenzhen), and participated in the creation of several start-up companies [ 53 ]. The next step in his career resulted in the biggest medical scandal of the last decade. In 2017 on WeChat social media platform, He announced that he was recruiting volunteers from among married couples who wanted to produce children genetically modified to be resistant to the human immunodeficiency virus (HIV). Among the conditions of recruitment was that in the couple who wished to participate in the experiment both people had a university degree, so that they had enough educational background to understand the basics of science and medicine. A second condition was for the man to be HIV-positive and for the woman – HIV-negative: a situation in which the risk of transmitting the virus to the baby would be minimal (provided that the sperm was “washed” during the IVF procedure), but made it likely that the couple’s motivation to participate in the experiment would be high [ 53 ]. He planned to modify the CCR5 gene, a known receptor on the cell surface, through binding to which the human immunodeficiency virus enters the cell. About 300 people responded to the advertisement, of these, 20 couples were selected for the next round of consultations, during which the participants learned about the procedure and the possible risks. From these consultations 11 couples agreed to participate in the studies, of which seven were ultimately selected by the researchers for the next stage – the IVF procedure with an additional step of genome editing. The motivation of individual participants was, apparently, not only the possibility of having children (the IVF procedure in China is prohibited if one of the parents has HIV infection), but also the desire to take part in an “historic” experiment designed to benefit future generations [ 53 ]. Ultimately, after several unsuccessful attempts, from a selected group of participants 2 pregnancies led to the birth of babies who had undergone a genomic modification procedure using the CRISPR–Cas9 system. Quite a lot is known about the first pregnancy, which resulted in the birth of two twin girls, Lulu and Nana (pseudonyms used in the press and scientific literature in order to protect their identity). Very little information is available on the second pregnancy, which resulted in the birth of another child. Since this event occured after the scandal caused by the birth of the first twins, many details of the second pregnancy remained a secret. A manuscript written by He, based on the results of the first pregnancy and named “Birth of twins after genome editing for HIV resistance” remains unpublished, but has been leaked to the scientific community [ 54 , 55 ]. It has become known, for example, that in one of the embryos both copies of CCR5 were inactivated (Nana), while in the second, only one was modified (Lulu) [ 56 ]. Therefore, only Nana has a chance to be protected from HIV infection in the future, at least from the main variants of the virus that enter the cell through binding to the CCR5 receptor. In the case of Lulu, unfortunately, the treatment will provide no protection, since one copy of the CCR5 gene is enough to produce the corresponding receptor on the membrane. It is believed that two embryos were implanted in the uterus of a future mother in the hope that at least one of them will lead to the birth of a genetically modification baby. The twins were born premature (at 31 weeks) and spent the first weeks of their lives in neonatal incubators but were otherwise described as “healthy” [ 53 ]. Scientists who had gained access to the unpublished manuscript of He, also noted that several cells selected for sequencing early in embryonic development were in fact mosaics, an observation that led to increased criticism of He’s work. In the case of mosaicism, any information obtained during the sequencing of selected cells cannot be extrapolated to the entire embryo as a whole. Therefore, at the time of the key decision of whether to transfer the embryos into the womb, the researchers could not be sure that the CRISPR–Cas9 system did not produce any dramatic off-target mutations in the remaining cells of the embryos, even if the sequencing results showed the absence of such modifications in the selected cells. Many other aspects of the conduct of the study also received harsh criticism from the scientific and medical community [ 54 ], including the questionable circumstances of obtaining permission from the ethics committee of a hospital in Shenzhen, the level of qualification of He for clinical research (lack of medical education and adequate experience in the field), the choice of the gene that has undergone editing (social rather than medical reasons for patients seeking help), possible side effects from the lack of a valid copy of CCR5 , etc. According to an American cardiologist and Professor of Medicine at the University of Pennsylvania Kiran Musunuru, the first babies of “the CRISPR generation”, unfortunately, were born not as a result “of a historic scientific achievement, but rather a historic ethical fiasco” [ 56 ]. A preceding PR-campaign conducted by He and his team resulted in fairly flattering initial news coverage of his work in the People’s Daily (the largest newspaper group in China). However, the following international scandal led to the placement of He under house arrest, and then to a 3-year prison sentence. He has already been released from prison, but little is known about his whereabouts and future plans [ 57 ].

A few months after the described scandal the Russian scientist Denis Rebrikov stirred up the international scientific community with a statement about his intention to become the second scientist in the world to create genetically modified babies. Rebrikov, a Professor at the Pirogov Russian National Research Medical University and Head of the Laboratory of Genomic Editing at the Center for Obstetrics, Gynecology and Perinatology, announced that his research facility was potentially ready to transfer modified embryos into the mother’s womb in June 2019 [ 58 ]. As in the experiments of He, he was planning to edit the CCR5 gene, and the preliminary work from his laboratory on non-viable embryos was published in the Bulletin of the Russian National Research Medical University [ 59 ]. The reaction of the scientific community to the statement was heated and primarily negative. In October 2019 the journals Nature and Science published news feeds reporting that at that time, Rebrikov had already switched to editing the GJB2 gene associated with inherited deafness, and was in the process of selecting couples who would agree to take part in the experiment [ 60 , 61 ]. However, in numerous interviews with journalists Rebrikov emphasized that he would only conduct such experiments after obtaining all necessary permits from both regulatory and ethical authorities. This significantly distinguished his approach from He’s, who informed the scientific community about the birth of babies with a modified genome post factum . The Ministry of Health of the Russian Federation (following the recommendation of the World Health Organisation) later made a statement that the decision to grant permission for such a study would be premature and irresponsible, which prevented the further development of the situation at least until the situation in the regulatory sphere changes [ 62 ].

At the time of writing this review, the state of the legal framework that regulates the issue of genomic editing of human embryonic cells varies greatly in different countries. Thus, genomic modification of embryos for purposes other than reproductive is allowed in at least 11 countries, including China, the U.S., and the U.K. Nineteen countries, including Belarus, Canada, Sweden, and Switzerland, prohibit such experiments. Many other countries (Russia among them) take an intermediate or indeterminate position. The situation with the introduction of inherited genomic changes into embryos subsequently used for reproductive purposes is even more complicated [ 63 ].

MEDICAL APPLICATIONS WITH HUMAN SOMATIC CELLS

Despite increased attention to the introduction of heritable changes in germline cells, the less controversial and currently more common use of CRISPR–Cas9 for medical purposes is the modification of human somatic cells. As described above, in the first attempts at gene therapy (1990) an adeno-associated viral vector was used that delivered a healthy copy of the gene into cells (in the U.S. this technology was finally approved for clinical use only in 2017 [ 64 ]). The next step in the development of gene therapy was the introduction of genomic editing with the use of Homing Endonucleases (HEs), Zinc Fingers Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and later also CRISPR–Cas9 [ 65 ]. The first human clinical studies using CRISPR–Cas9 commenced in October 2016 in China [ 66 ]. The PD-1 gene was inactivated ex vivo in blood cells in the hope that such modified cells, would attack the non-small-cell lung cancer that the patient suffered from when returned to circulation. In the U.S., ex vivo therapy using CRISPR–Cas9 was first performed in July 2019 on a patient with sickle cell anemia (CRISPR Therapeutics, founded by Charpentier). The therapy significantly improved the patient’s condition for at least a few months after the procedure, however the cost of such treatment at the time of its implementation in the United States was estimated to be in the region of 0.5-1.5 million U.S. dollars. The high current cost of CRISPR–Cas9 therapy will probably act as an obstacle to its widescale use, even if clinical trials confirm the efficacy and safety of such treatment [ 18 ]. Currently, the most expensive drug on the market is Zolgensma, another gene therapy treatment used for spinal muscular atrophy ($2.125 million per dose). Zolgensma directly delivers a working copy of the defective gene into cells with the use of adeno-associated virus, a method different from genomic editing using nucleases [ 67 ].

The first example of an in vivo clinical study in which cells undergo in situ genomic editing with nucleases was performed using the ZFNs technology. Sangamo Therapeutics first performed this procedure in July 2017 on a patient suffering from Hunter syndrome (a rare genetic disease, form of mucopolysaccharidosis). The pioneers in using CRISPR–Cas9 for in vivo genomic editing were Editas Medicine (March 2020) [ 68 ]. A drug called EDIT-101 was injected locally into the retina of a patient suffering from a form of inherited blindness caused by a mutation in the CEP290 gene. Currently, various clinical studies are underway on the use of CRISPR–Cas9 for the treatment of diseases such as Alzheimer’s disease, various types of cancers, high cholesterol, angioedema, acute myeloid leukemia, and even androgenetic alopecia (baldness). Another promising application for CRISPR–Cas9 in the future could be the treatment of infectious diseases caused by such pathogens as, for example, HIV and human papillomavirus [ 65 ].

CONCLUSIONS

The discovery of CRISPR–Cas9 as an immune system in prokaryotes at the turn of the 20th-21st centuries – a finding at first glance only relevant to microbiology – has led to a revolution in the field of genomic manipulations. New opportunities have opened up in multiple areas of biomedicine, such as molecular diagnostics of infectious and non-infectious diseases (e.g., genotyping of bacterial strains, detection of viruses, and identification of genetic mutations in circulating extracellular DNA in patients with lung cancer [ 69 ]), as well as in the development of a potentially new method of immunization, DNA vaccines [ 18 ]. One of the more unusual examples of the application of the CRISPR–Cas9 system was the cultivation of brain-like organelles carrying different variants of the important NOVA1 gene characteristic of modern humans, Neanderthals, and Denisovans [ 70 ]. The development of CRISPR–Cas9 technology is a good example of how discoveries made in the course of basic research can change entire fields of science and technology, expanding the horizons of the possible. This ground-breaking technique is a worthy continuation of such exciting scientific events as the publication of the double-stranded structure of DNA by Watson and Crick in 1953, the birth of the first child by in vitro fertilization in 1978, and the cloning of Dolly the sheep in 1996. In the coming years the scientific community will watch with interest the development of legislation and ethical principles in the application of the CRISPR–Cas9 system in genome editing, as well as in what other areas of science this promising technology will find its application.

Abbreviations

CRISPR-associated genes

clustered regularly interspaced short palindromic repeats

CRISPR-associated RNA

protospacer adjacent motif

single guide RNA

Cas9 protein from Streptococcus pyogenes

trans-activating CRISPR RNA

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Acknowledgments

The author recalls with warmth and gratitude the years spent in the laboratory of Andrei Dmitrievich Vinogradov at the Department of Biochemistry of Moscow State University. The experiments conceived by Andrei Dmitrievich invariably brought interesting results, while his vast knowledge in various fields of science enabled staff and students to feel confident that any questions would be answered, and the time spent in the laboratory would bring well-deserved results. The publication of the results of the work carried out under the supervision of Andrei Dmitrievich gave the author the necessary start in scientific life and the opportunity to continue research in other laboratories and other fields of knowledge. A unique team of scientists, selected by Andrei Dmitrievich: Vera Georgievna Grivennikova, Tatiana Vadimovna Zharova, and Eleonora Vladimirovna Gavrikova, provided a family atmosphere of trust and support in the laboratory, for which the author is very grateful.

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Gostimskaya, I. CRISPR–Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing. Biochemistry Moscow 87 , 777–788 (2022). https://doi.org/10.1134/S0006297922080090

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Received : 11 May 2022

Revised : 07 July 2022

Accepted : 19 July 2022

Published : 15 August 2022

Issue Date : August 2022

DOI : https://doi.org/10.1134/S0006297922080090

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