CRISPR-Cas9, or just CRISPR Modern gene editing tech Derived from a primitive immune system in certain bacteria Cas9 is a crucial CRISPR protein; it cleaves DNA at a specified point
1987: Discovered the repeats 2007: CRISPR might be an immune system 2010: Cas9 is guided by RNA and cleaves DNA 2013: Edited eukaryotic cells with CRISPR
DNA, proteins, etc, normally can’t enter bacterial cells, a transformation mix makes them “competent” or receptive to DNA and whatnot.
IV: Composition of transformation mix DV: Transformation efficiency CVs: Agar contents Sterile technique Amount of transformation mix used Amount of bacteria spread on plates
CaCl2: Neutralize DNA’s negative charge PEG: Neutralize DNA’s negative charge, make cell membrane more porous, maybe aid in DNA transport DMSO: Make cell wall more permeable, loosen DNA structures
Pour plates and number them
Plate bacteria and check for proper resistances
Mix transformation mix and some bacteria
Mix competent cells and DNA stuffs, heat shock and add to an LB tube
Incubate and plate final CRISPR mixtures
Things didn’t go so well…!
CRISPR bacterial transformation mixes
in CRISPR-Cas9 Systems
Lakeland Jr-Sr High School
What 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.
(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
As 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.
The experimental transformation
mix will increase transformation
efficiency, therefore rendering
more bacteria competent and
resulting in more bacterial growth
compared to the standard mix.
• Composition of
•Amount of transformation mix used
•Amount of bacteria spread on plates
25mM 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).
Adapted from The ODIN’s DIY CRISPR Kit (The ODIN, 2016)
Equipment Misc Perishables
• 1 - 100uL variable volume
*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
• 6x Petri plate
• 1 LB Agar (6g in a 15mL tube)
• 1 LB Strep/Kan Agar (6g in a
• 1 250 mL glass bottle for pouring
plates (fill with 100- 150mL
• 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
• crRNA plasmid, 55uL of
• Template DNA, 55uL of 100ng/uL
Setup, create competent cells, and create CRISPR mixture
Amended 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
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
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
1. 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 SHOULD be present on the LB plate.
Growth SHOULD NOT be present on the LB + Strep + Kan plate.
Create competent cell mixture
Perform 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
2. 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
4. Label tube appropriately.
"Ctrl." for the standard transformation mix
"Exp." for the experimental transformation mix
5. Store at 4C
Create CRISPR mix
1. Add 10uL of Cas9 and tracrRNA to the competent cell mixture
2. Add 10uL of crRNA to the competent cell mixture
3. Add 10uL of template DNA to the competent cell mixture
4. Store in fridge for 30 minutes
5. Heat shock cell mixture tube for 30 seconds in 42C water
6. Add 1.5mL water to an LB media microcentrifuge tube and shake
7. Add 500uL of LB media to the competent cell mixture
8. Label tube appropriately.
a. "Ctrl." for the standard CRISPR mix
b. "Exp." for the experimental CRISPR mix
Create CRISPR mix cont.
9. Incubate at 37C for 2 - 4 hours
10. 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°C
12. Check for growth, see if there is any difference between growth in the
control and experimental groups.
Results, Sources of error and Conclusion
= Agar has Streptomycin +
= Competent cells (made
with control transformation mix)
= 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,
6, LB + Streptomycin + Kanamycin,
All plates had growth on them.
All LB + strep + kan plates had less growth than their antibiotic-free
On the LB, antibiotic-free plates, the control plate had more growth than the
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
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
Due to multiple sources of error and a limited number of trials, no conclusions
can be drawn.
Run the experiment again with multiple trials
Make agar plates without pre-mixed antibiotics to reduce the risk of heat-
inactivating the antibiotics
Patrick D. Hsu, E. S. (2014, June 5). Development and Applications of CRISPR-Cas9 for
Genome Engineering. Retrieved from PubMed:
The ODIN. (n.d.). DIY CRISPR Genome Engineering. Retrieved from The ODIN: