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Introduction

CRISPR is an acronym of 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 to 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, 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 modify the genomic DNA at home. It only requires work of 10 hours over the spread of at least 2 days.

Supply List

LB Agar
Quick Dissolve, Clear Background, Enriched Nutrients For Better Bacterial Growth
Inoculation Loops/Plate Spreader
INOCULATING LOOP Consists of a 3`` long, 25 gauge Nichrome wire with a loop at one end and the other end mounted in an 8`` long aluminum handle. 3/pk
10-100uL variable volume adjustable pipette
10-100uL variable volume adjustable pipette (1uL increments)
Box of 96 Pipette Tips
The tip is changed every time a pipette is used
14 Petri Plates
Used to spread the mixture in and grow bacteria
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Micro-centrifuge tube rack
Holds the micro-centrifuge tube
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Nitrile Gloves
Gloves for handling
PEG 8000 5% DMSO
PEG 8000 5% DMSO
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Micro-centrifuge tubes
It is used to keep the transformation mix
50mL Tube
For measuring

 Supplies needed:

  • Cas9 and tracrRNA plasmid
  • crRNA plasmid
  • Template DNA
  • Preparation Time:
    • 1 hour to make plates
    • 1 Minute to streak out bacteria
    • 12-18 hours to grow the bacteria

    If you are making plates for the first time, you will probably need more than an hour. To grow bacteria, it is advisable to let it sit overnight.

On the day of Experiment:

  • 5 minutes to mix the sample, plasmids and transformation mix
  • Refrigerate for 30 minutes, but do not freeze
  • Heat shock the mixture for 30 seconds
  • Add LB media to cell solution. This will roughly take 1 minute
  • Incubate the solution for 1-2 hours at 30 C or at least for 4 hours at room temperature
  • Plate the 200 uL of the solution and let it dry for 10 minutes.

Incubation Period:

Incubate for 16-24 hours at 30ºC (86ºF) or at room temperature for 24-48 hours.

Experiment Protocol:

A step by step guide to for this DIY Crispr experiment at home is as follows

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Nitrile Gloves
Nitrile Gloves can be easily purchased separately online. These are disposable puncture resistance gloves, specially designed for medical-related services, food handling, parts handling, janitorial services, painting and finishing services.

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 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 it into a 250 mL glass bottle. LB Agar is used for growing micro-organisms including bacteria, yeast, molds, etc.

LB Agar
LB Agar is used for growing micro-organisms including bacteria, yeast, molds, etc.

Measure 150 mL of water using 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 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 in 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’s better to leave the plates out for a couple of hours or overnight so that compression doesn’t affect the plates.

Step 3:

Make Competent Bacterial Cells for Transformation

Competent cells are those bacteria or yeast cells that intake foreign DNA. Typically, 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.

Inoculation Loop
Inoculation Loop

Use the inoculation loop to put bacteria on it and then gently smudge it on the plate. Inoculation loops are used to transfer micro-organism 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 plate is put in 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.

Now make the bacterial transformation mixture by adding:

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 cell membrane more absorbent. Just like PEG 3350, CaCl2 also neutralize the negative charge of DNA and makes it easier to permeate the cell.

Mix PEG and CaCl2 together in quantity mentioned above to make the mixture. Now use 100 uL pipette to fill the micro-centrifuge tube with transformation mix. A pipette is used to dispense volumes of liquid accurately and safely.

BasicPette Multi-Volume Adjustable and Fixed Volume MicroPipette / Pipettor
BasicPette Multi-Volume Adjustable and Fixed Volume MicroPipette / Pipettor

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 full 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.

Main parts of CRISPR

CRISPR has three main parts:

  • Cas9 Protein
  • guide RNA (gRNA)
  • Template DNA

Cas9 Protein

This is the most important part of CRISPR and serves at its engine. Its job is to bind together gRNA and also targets 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.

Guide RNA (gRNA)

The gRNA is composed of CRISPR RNA (tracrRNA) and the CRISPR RNA (crRNA) and connected by small nucleotide linker. You can also use these separately for the experiment, but it is better to use gRNA that is available.

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.

Template DNA

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 true 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 to produce a code for Lysine instead of Threonine in an important protein. It also prevents Streptomycin from binding to and disabling the protein hence, resulting in the bacteria cell to grow on media containing it.

Steps of the CRISPR Reaction

  1. Cas9 binds the gRNA molecule
  2. gRNA matching DNA sequence is found

iii.    Cas9 cuts the DNA

  1. Cell tries to repair the break using DNA template
  2. Cell gets a new DNA sequence

DNA Transformation & CRISPR Experiment

  1. Add 10uL of Case9 and TracrRNA in the competent cell mixture using a pipette. Change the pipette tip with every use.
  2. Add Template DNA with the help of pipette to the same mixture.
  3. Put the tube in the fridge for 30 minutes, make sure it doesn’t freeze. Then leave it in warm water at a temperature of 42ºC (108ºF) for 30 seconds.
  4. Add 500 uL of LB media to the mixture using the pipette.
  5. Again incubate the tube at 30ºC (86ºF) for 2 hours. If you want to keep it at room temperature, then leave it for 4 hours.
  6. Now add 1.5 mL of room temperature water to one of the LB media micro-centrifuge tubes and shake it well to dissolve the LB. These micro-centrifuge tubes ensure safety while testing any samples and materials. After that, add 500 uL of LB media in the competent mixture using the pipette.

Micro-Centrifuge Tube: https://www.amazon.com/1-5ml-Microcentrifuge-Polypropylene-Graduated-Natural/dp/B01C4SSDJU/ref=sr_1_4?s=industrial&ie=UTF8&qid=1513149146&sr=1-4&keywords=%E2%80%A2%09Micro-centrifuge+tubes

 

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Micro-Centrifuge Tube
Micro-Centrifuge Tube
  1. Incubate for 2-4 hours at 30ºC (86ºF). This is the key step in the experiment, therefore, don’t skimp on the time. If you face any trouble, you can increase the incubation time up to 12 hours. Now take the LB/Strep/Kan/Arab plate out of the fridge and let it warm at room temperature.
  2. Spread the CRISPR transformation mixture on top of LB Strep/Kan/Arab Agar plate.
  3. Use an inoculation tube to spread the bacteria around the plate and leave it to dry for at least 10 minutes. After that put its lid back on the plate.
  4. In order to prevent condensation of bacteria, flip the plate upside down.
  5. Incubate the plate for 16-24 hours at 30ºC (86ºF) or 24-48 hours at room temperature.
  6. Small white dots start growing on the plate will signal the success of your experiment. If not, you can give it another try.

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.

Author Conductscience

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