Effectiveness of Different Bacterial Transformation Mixes in CRISPR-Cas9 SystemsJohn KozloskyLakeland Jr-Sr High School

IntroductionWhat is CRISPR-Cas9, and why do we need a transformation mix to use it?

What is CRISPR-Cas9?Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a modern gene editing technology derived from a primitive viral immune system present in certain bacteria. CRISPR Associated Protein 9 (Cas9), an RNA-guided DNA endonuclease, is at the heart of the CRISPR-Cas9 system. CRISPR is actually used by bacteria to store bits of DNA from viruses that previously invaded the cell, and send out Cas9 proteins to watch for any instances of that viral DNA or RNA and inactivate it upon detection by snipping the strand of DNA or RNA.

However, scientists do not use CRISPR for this reason – they use the Cas9 endonuclease to make precise edits of DNA where they desire, which allows for insertion, removal, or modification of genes. Scientists essentially use CRISPR as a tool for genetic engineering.

CRISPR Timeline(Patrick D. Hsu, Eric S. Lander, and Feng Zhang, 2014)

Why do we need a transformation mix?In order for the required DNA, RNA, proteins, etc, to enter bacterial cells and allow Cas9 edit their DNA, the bacteria must be modified, or made competent. This is because under normal circumstances, the bacterium’s thick, negatively charged cell wall repels the negatively charged DNA, RNA, and other chemicals, rendering them unable to pass into the cell.

However, not all hope is lost – through a few special steps and a solution called a transformation mix, the materials we want inside the bacterium can be forced into the cell.

What is in the transformation mix?Control mix: 25mM Calcium Chloride (CaCl2)Experimental mix: 25mM CaCl2, 10% PEG 8000, and 5% DMSO

PurposeAs CRISPR is a relatively new tool, the most effective methods for using it properly have not yet been discovered or developed. Determining better parameters for increasing transformation efficiency will allow scientists to use CRISPR faster and more effectively.

HypothesisThe experimental transformation mix will increase transformation efficiency, therefore rendering more bacteria competent and resulting in more bacterial growth compared to the standard mix.

Independent Variable

• Composition of transformation mix

Dependent

Variable• Transformatio

n efficiency

Control Variables

• Agar contents • Sterile technique • Amount of transformation mix used• Amount of bacteria spread on

plates

Background Research25mM CaCl2 (Calcium Chloride): CaCl2 is thought to shield or neutralize the negative charge of the DNA, therefore making it more likely to enter the negatively charged cell wall (The ODIN, 2016).10% PEG 3350 (Polyethylene Glycol): PEG is thought to shield DNA’s negative charge, make the cell membrane more porous, and perhaps aid in transporting the DNA into the cell (The ODIN, 2016).5% DMSO (Dimethyl Sulfoxide): DMSO may make the cell wall more permeable and loosen DNA if it folds into complex structures (The ODIN, 2016).

MaterialsAdapted from The ODIN’s DIY CRISPR Kit (The ODIN, 2016)

Equipment Misc Perishables• Incubator• 1 - 100uL variable volume

Micropipette

*Note: These materials may actually be more than required to complete one round of experimentation – however, this is simply the list of materials included in The ODIN’s CRISPR kit.

• 6x Petri plate• 1 LB Agar (6g in a 15mL tube)

  • 1 LB Strep/Kan Agar (6g in a

15mL tube)    • 1 250 mL glass bottle for

pouring plates (fill with 100- 150mL water)

• 1 Microcentrifuge tube rack  • 5 1.5mL microfuge tubes

containing  .03g LB   • 50mL centrifuge tubes• 1 mL bacterial transformation

buffer (25mM CaCl2, 10% PEG 3350 5% DMSO)

• 1mL bacterial transformation buffer (25mM CaCl2)

• Inoculation loops

• E. coli HME63 strain• Cas9 and tracrRNA plasmid,

55uL of 100ng/uL     • crRNA plasmid, 55uL of

100ng/uL    • Template DNA, 55uL of

100ng/uL

ProcedureSetup, create competent cells, and create CRISPR mixture

SetupAmended from The ODIN’s DIY CRISPR kit instructions (The ODIN, 2016)1. Pour plates

3x LB agar 3x LB agar with Streptomycin and Kanamycin antibiotics ~25mL water and 4g agar per plate

2. Label the LB agar plates "LB" and the LB agar with Strep and Kan plates "LB + Strep + Kan."

3. Number the plates from 1 - 6 according to the diagram below.4. Write "Test" after your labels on plates 1 and 4. These plates will be used to test

if the bacteria have the proper antibiotic resistances or not.5. Write "CRISPR Ctrl." after your labels on plates 2 and 5. These plates will

contain the CRISPR-modified bacteria using the standard/control bacterial transformation mix.

6. Write "CRISPR Exp." after the labels on plates 3 and 6. These plates will contain the CRISPR-modified bacteria using the experimental bacterial transformation mix.

SetupPlate Labeling Diagram

1 LB (Test) 2 LB (CRISPR Ctrl.) 3 LB (CRISPR Exp.)

4 LB + Strep + Kan (Test)

5 LB + Strep + Kan (CRISPR Ctrl.)

6 LB + Strep + Kan (CRISPR Exp.)

SetupResistance check1. Get plates 1 "LB (Test)" and 4 "LB + Strep + Kan (Test)."2. Plate the bacteria onto each plate.3. Flip plate upside down and incubate at 37°C for 12 – 18 hours and

wait for growth. Growth SHOULD be present on the LB plate. Growth SHOULD NOT be present on the LB + Strep + Kan plate.

Create competent cell mixturePerform these steps once with the standard transformation mix and once with the experimental transformation mix.1. Pipette 100uL of the appropriate transformation mix into a

microcentrifuge tube2. Scrape off some bacteria from plate "1 LB (Test)" onto an inoculation

loop and mix into the transformation mix in the microcentrifuge tube The mixture should be cloudy, if not, mix in more bacteria

3. Mix4. Label tube appropriately.

"Ctrl." for the standard transformation mix "Exp." for the experimental transformation mix

5. Store at 4C

Create CRISPR mix1. Add 10uL of Cas9 and tracrRNA to the competent cell mixture2. Add 10uL of crRNA to the competent cell mixture3. Add 10uL of template DNA to the competent cell mixture4. Store in fridge for 30 minutes5. Heat shock cell mixture tube for 30 seconds in 42C water6. Add 1.5mL water to an LB media microcentrifuge tube and shake7. Add 500uL of LB media to the competent cell mixture8. Label tube appropriately.

a. "Ctrl." for the standard CRISPR mixb. "Exp." for the experimental CRISPR mix

Create CRISPR mix cont.9. Incubate at 37C for 2 - 4 hours10. Add 200uL of CRISPR transformation mixture (what you just made) to

an LB plate and another 200uL to an LB + Strep + Kan plate. Spread bacteria across plate gently. Let dry for 10 minutes.

1. Use plates 2 and 5 for the standard CRISPR mixture.2. Use plates 3 and 6 for the experimental CRISPR mixture.

11. Flip plate upside down and incubate for 1 day at 37°C12. Check for growth, see if there is any difference between growth in the

control and experimental groups.

ResultsResults, Sources of error and Conclusion

Results = Agar has Streptomycin + Kanamycin antibiotics

= Competent cells (made with control transformation mix) + CRISPR

= Competent cells (made with experimental transformation mix) + CRISPR

1, LB, “Test,” Not competent 2, LB, “CRISPR,” Control 3, LB, “CRISPR,” Experimental

4, LB + Streptomycin + Kanamycin, “Test,” Not competent

5, LB + Streptomycin + Kanamycin, “CRISPR,” Control

6, LB + Streptomycin + Kanamycin, “CRISPR,” Experimental

ResultsAll plates had growth on them.All LB + strep + kan plates had less growth than their antibiotic-free counterparts.On the LB, antibiotic-free plates, the control plate had more growth than the experimental plate.On the LB + strep + kan plates, the experimental plate had slightly more growth than the control plate.

Sources of Error Contamination: Agar plates are highly susceptible to contamination,

which may alter the outcome of the experiment. Ways to reduce contamination: Autoclave materials and practice better

sterile technique Limited number of trials: Only one trial was completed

Solution: Run more trials Antibiotic inactivation: Streptomycin and Kanamycin antibiotics may

have been partially or wholly inactivated while agar was being microwaved – may explain why antibiotic plates still exhibited growth Solution: Use agar that does not contain pre-mixed antibiotics; Mix

antibiotics in after agar has been fully melted

ConclusionDue to multiple sources of error and a limited number of trials, no conclusions can be drawn.

Further Studies Run the experiment again with multiple trials Make agar plates without pre-mixed antibiotics to reduce the risk of

heat-inactivating the antibiotics

Works CitedPatrick D. Hsu, E. S. (2014, June 5). Development and Applications of CRISPR-Cas9 for

Genome Engineering. Retrieved from PubMed:https://www.ncbi.nlm.nih.gov/pubmed/24906146

The ODIN. (n.d.). DIY CRISPR Genome Engineering. Retrieved from The ODIN:http://www.the-odin.com/crispr-bacterial-guide/