Genome-scale CRISPR Knock-Out (GeCKO) Library Transformation
Introduction
Materials
- GeCKO Library (Addgene)
- Electrocompetent cells (NEB)
- Maxiprep Kit (Qiagen)
- HEK293(F)T cells (Invitrogen)
- Packaging plasmids
- lentiCRISPRv2 and lentiGuide-Puro backbone vectors are compatible with both 2nd and 3rd generation lentiviral packaging plasmids (see Addgene).
Procedure
- Dilute the GeCKO library to 50ng/µL in water or TE (if not already diluted).
- Electroporate the library.
Add 2µL of 50ng/µL GeCKO library to 25µL of electrocompetent cells with an efficiency of ≥ 10^9 cfu/ug. We have had success with many electrocompetent cells, including NEB DH5a cells, Invitrogen DH5a cells and Lucigen Endura. In our hands, the Lucigen Endura cells (cat # 60242) have yielded the highest efficiency with the GeCKO library.
Electroporate using the manufacturer’s suggested parameters/protocol.
Recover in 975µL recovery media (i.e. media provided with cells) and transfer to a loosely capped tube with an additional 1mL of recovery media.
Repeat for a total of 4 electroporations and rotate at 250 rpm for 1 hour at 37 C.
- Plate a dilution to calculate transformation efficiency.
Note the library plasmids have ampicillin resistance – prepare all plates accordingly.
Pool all 8mL of electroporated cells. Mix well.
Remove 10µL and add to 1mL of recovery media, mix well, and plate 20µL onto a pre-warmed 10cm petri dish (ampicillin). This is a 40,000-fold dilution of the full transformation and will enable you to estimate transformation efficiency to ensure that full library representation is preserved.
- Plate the transformations.
Follow Step a) if your lab has 24.5 cm^2 bioassay plates for large-scale bacteria culture; otherwise follow Step b), which substitutes 20 standard (10 cm round) petri dishes.
a) Plate 4mL of transformation on each of 2 pre-warmed 24.5 cm2 bioassay plates (ampicillin) using a spreader. Spread the liquid culture until it is largely absorbed into the agar and won’t drip when turned upside down.
b) Alternatively, spread 400µL of transformation mix per petri onto 20 pre-warmed petri dishes.
- Grow all plates inverted for 14 hours at 32 C.
Growth at this lower temperature reduces recombination between the lentiviral long-terminal repeats. (Growth at 37 C is also acceptable if 32 C is not possible.)
- Calculate transformation efficiency.
Count the number of colonies on the dilution plate.
Multiple this number of colonies by 40,000 for the total number of colonies on all plates.
Proceed if the total number of colonies is at least 3 × 106. This efficiency is equivalent to 50X colonies per construct in the GeCKO library (i.e. 50X for library A or B individually).
- Harvest colonies.
Pipette 10mL of LB onto each 24.5 cm^2 bioassay plate (or, 500µL per 10 cm petri dish).
Scrape the colonies off with a cell spreader/scraper.
Pipette off the liquid plus scraped colonies into a tube and repeat the procedure a second time on the same plate with additional 5-10mL. Note: Weigh this tube prior to adding any liquid to it.
- Weigh the bacterial pellet to determine the proper number of maxiprep columns to use.
Spin down all liquid to pellet the bacteria and then discard the supernatant.
Weigh the bacterial pellet and subtract the weight of the tube.
- Maxi-prep for downstream virus production and future amplification.
Using a maxi scale plasmid prep, each column can handle approximately 0.45g of bacterial pellet.
Perform a sufficient number of maxi preps so as to not overload a column.
- Proceed to transient transfection of HEK293(F)T cells with maxi-prepped GeCKO lentiCRISPR library and appropriate packaging plasmids.
If producing virus from the full library, transfect cells with both A and B half-library plasmids.
Library plasmid should also be deep sequenced to verify representation and pool complexity.
GeCKO v2.0 library specifications
A A | B B | C C | |
1 1 | GeCKO v2 human library | GeCKO v2 mouse library | |
2 2 | Species | human | mouse |
3 3 | Number of genes targeted | 19,050 | 20,611 |
4 4 | Targeting constructs per gene | 6 per gene (3 in Library A, 3 in Library B) | 6 per gene (3 in Library A, 3 in Library B) |
5 5 | Number of miRNA targeted | 1,864 | 1,175 |
6 6 | Targeting constructs per miRNA | 4 per miRNA | 4 per miRNA |
7 7 | Control (non-targeting) sgRNAs | 1,000 | 1,000 |
8 8 | Total sgRNA constructs | 122,411 (65,383 in Library A, 58,028 in Library B) | 130,209 (67,405 in Library A, 62,804 in Library B) |
9 9 | Viral plasmid vector | Single and dual vector: lentiCRISPR v2 and lentiGuide-Puro | Single and dual vector: lentiCRISPR v2 and lentiGuide-Puro |
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a microbial nuclease system containing a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage. Using lentivirus, we delivered the type II CRISPR nuclease system to facilitate genome editing in mammalian cells in a pooled library targeting early consecutive exons (Shalem, Sanjana, et al., Science 2014). Here we describe how to amplify GeCKO v2.0 DNA plasmids to have sufficient quantity to produce lentivirus, while maintaining full library representation. For further background, see information from the Zhang Lab. Also see the GeCKO website and the CRISPR forum for any help needed.