CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats which are an important part of the bacterial defense system and form the bases for CRISPR-Cas9 genome editing technology. This system is programmed to target specific genetic codes and edit DNA. It is also used for various other purposes as well including new diagnostic tools. These systems have enabled researchers to modify the genetic codes of living organisms permanently. It is expected that in the near future they will also help in treating genetic disorders by correcting specific mutations in the human genome.
A Spanish scientist named Francisco Mojica was the one who first discovered CRISPRs in archaea and later in bacteria. He propagated that CRISPRs serve as part of the bacterial immune system that defends against viruses. In 2007, a team led by Philippe Horvath experimentally demonstrated Mojica’s theory. While in 2013, the Zhang lab successfully published the first method to engineer CRISPR to edit the genome in mouse and human cells.
Following is the easy-to-use CRISPR Cas9 system guide to modifying the genomic DNA at home. It only requires work of 10 hours over the spread of at least 2 days.
Incubate for 16-24 hours at 30ºC (86ºF) or at room temperature for 24-48 hours.
A step by step guide for this DIY Crispr experiment at home is as follows
Step 1:
Put on a pair of Nitrile Gloves
Before doing any sensitive experiment, it is very important to take all the precautionary measures. This experiment involves growing bacteria and yeast and our skin act as a natural host for these, therefore it is necessary to wear gloves.
These are disposable puncture resistance gloves, specially designed for medical-related services, food handling, parts handling, janitorial services, painting, and finishing services.
Take out LB Agar and LB Strep/Kan/Arab Agar Media and pour them into a 250 ml glass bottle. LB Agar is used for growing micro-organisms including bacteria, yeast, molds, etc.
Measure 150 mL of water using a 50 ml conical tube and add it to the glass bottle. Heat the bottle for 30 seconds at a time in a microwave and make sure that the lid is not screwed tight. Just place it on top of the glass bottle. When the liquid turns yellow, it means that it’s done. This will take around 2-3 minutes in total of microwaving. Wait for 20-30 minutes to let it cool down. Now take out your plates and fill them one at a time. Pour out the solution while the bottle is still warm. Pour just enough LB Agar to cover the bottom of the plate. This will help to make a maximum number of Agar plates. Close the lid of the bottle after pouring the solution into the plates. Let it cool for at least an hour before use. You can also cool it down in the fridge but don’t freeze it. It’s better to leave the plates out for a couple of hours or overnight so that compression doesn’t affect the plates.
Step 2:
Make Competent Bacterial Cells for Transformation
Competent cells are those bacteria or yeast cells that intake foreign DNA. Typically, the cell wall prevents foreign material from entering, but we will mix the bacteria with chemicals that will change this. Through the process of transformation, we need all the components to enter the cells to get CRISPR to work. The trick is to make bacteria believe that our synthetic DNA is its own DNA, so they make the Cas9 protein, the tracrRNA, and crRN.
Use the inoculation loop to put bacteria on it and then gently smudge it on the plate. Inoculation loops are used to transfer micro-organisms to growth media. These are easy to use and can be sterilized for reuse. Leave the plate overnight or for 12-18 hours to let the bacteria grow. The bacteria will be whitish and clearly visible on the plate. Make sure that the plate is put at a warm and consistent temperature. The cold place will slow down the bacterial growth process. You can save your bacteria for a week if you put it in a fridge, but it is more advisable to use fresh bacteria for the success of the experiment.
PEG 3350 plays an important role in the transformation process. Both DNA and cell walls are negatively charged, so they reject each other. PEG 3350 functions to shield DNA charge and make it easier to infiltrate the cell wall. It also helps transport DNA into the cell and makes the cell membrane more absorbent. Just like PEG 3350, CaCl2 also neutralizes the negative charge of DNA and makes it easier to permeate the cell.
Mix PEG and CaCl2 together in the quantity mentioned above to make the mixture. Now use a 100 uL pipette to fill the micro-centrifuge tube with a transformation mix. A pipette is used to dispense volumes of liquid accurately and safely.
Now use the inoculation loop to scrape some bacteria off of a fresh plate and then add it to the transformation mix. Make sure to fill the whole loop and gently mix it with the transformation mix until it is properly dissolved and there are no clumps. When the mixture turns cloudy, it means it’s done.
Make one tube for each CRISPR experiment you want to conduct and store them for 4ºC (39ºF) in the refrigerator if you want to conduct your experiment in a day or two. It is more advisable to plan one experiment at a time as this will you more opportunities to perform your experiment with more caution and for a number of times. You can also make two or three tubes to perform a single experiment.
CRISPR has three main parts:
This is the most important part of CRISPR and serves as its engine. Its job is to bind together gRNA and also target the gene for editing. Cas9 protein cuts the DNA when a gene match is found. The cell in return tries to repair the DNA.Cas9 doesn’t edit the gene in reality rather it tricks the cell into doing it.
The gRNA is composed of CRISPR RNA (tracrRNA) and the CRISPR RNA (crRNA) and connected by a small nucleotide linker. You can also use these separately for the experiment, but it is better to use the available gRNA.
The gRNA serves two functions. Its TracrRNA binds to Cas9 protein crRNA whereas, crRNA matches the DNA in the genome that we want to edit. It helps Cas9 protein to recognize the gene to cut.
Once the cell starts to repair DNA, the cells start looking for template DNA to figure out how to fill the gene. When we flood the cell with template DNA, the cell mistakes it for the actual copy and uses this instead. Our template DNA is made up of a single base change from an Adenine (“A”) to a Cytosine (“C”). This change results in DNA producing a code for Lysine instead of Threonine is an important protein. It also prevents Streptomycin from binding to and disabling the protein, resulting in the bacteria cell growing on media containing it.
The whitish or yellowish bacteria growing on the plate are the bacterial colonies that are successfully edited. This means that they have successfully survived and replicated.
Genetic engineering is one of the most useful developments in the field of science that has enabled humankind to deliberately modify the DNA of an organism and change any aspect of the organism for a particular purpose. This CRISPR experiment is a powerful way to modify genome DNA. It is easily available online has opened many opportunities for young scientists to take part in scientific learning. It enables users to learn many new concepts and technical skills like how to make agar plates, handle a pipette as well as the theory behind actual happening in the tubes.
Note: This is not a comprehensive guide rather it is just to show people how easy it is to c0nduct CRISPR-Cas 9 experiment at home for genome modification. If you are interested, you can go through various details about the history and procedures or CRISPR before starting your own experiment.
The bacteria supplied are not dangerous in any way since they are non-pathogenic and cannot cause any disease. You can easily dispose of by putting 5 % bleach on the plate and then throwing it away in the trash.
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