Creating pooled CRISPR-Cas9 knock-outs in NIH-3T3 cells

Generate 3T3-Cas9 cells

Infect NIH3T3-flpin cells with Cas9-containing lentivirus to generate cells constitutively expressing Cas9 as follows:

1. Plate NIH-3T3 cells into a 6 well cell dish at 1x105 cells / well
2. After cells attach, add lentivirus made from Lenti-Cas9 vector onto the cells (dx.doi.org/10.17504/protocols.io.bp2l61z2zvqe/v1)
3. After 48h 0m 0s, trypsinize cells and plate into 3 wells of a 6 well plate in media containing 10µg/ml Blasticidin
4. Cells infected with Lenti-Cas9 will survive Blasticidin and 72h 0m 0safter treatment, selection is stopped and Blasticidin-resistant survivors are expanded in normal medium

• Cas9 is toxic to these cells and they grow slower than the parental cells; These cells are more sensitive to cell death than usual and care must be taken to passage them regularly at 80% confluency. Change medium every 48 hours and avoid high passage numbers

1. At this point, test cells for Cas9 expression by western blot using mouse anti-Cas9 antibody (Biolegend 844302)
2. Sort 3T3-Cas9 cells to obtain single cells in a 96 well plate using a cell sorter; Allow cells to grow for 3 weeks with occasional medium exchange

• Cells that are grown in 25% conditioned medium (previous 3-day old 3T3 medium passed through a 0.2µm filter) grow faster
• Glutamax instead of Glutamine also helps cells grow faster
• Transfer clones that grow into 24 well plates and a few days later, into 6 well plates
• Expand each clone and freeze cells in liquid nitrogen

Identify the best 3T3 Cas9 clones

Healthy clones appear fibroblast-like, without vacuoles, and grow at a rate such that they need a 1:5 split every 3 days. Expand such clones and test for Cas9 expression by lysing in RIPA buffer and performing a western blot using anti-Cas9 antibody

Retain cells expressing the highest Cas9 and marker of choice (in our case, phospho-Rab10); of those, select clones that have the best growth and morphology

Test the top two clones for Cas9 activity by introducing a validated sgRNA known to produce knock-outs efficiently as follows:

1. Plate each 3T3 cell clone in wells of a 6 well plate (as above)
2. Infect cells 8 h after plating with a lentivirus carrying sgRNA targeting mouse GRK2 (Pusapati et al. 2018) that is known to knock-out GRK2 efficiently; perform an additional mock infection as a control
3. After , remove the virus supernatant, trypsinize cells and split cells into 2 wells of a 6 well plate and culture with 1µg/ml Puromycin. Cells not infected with the gRNA lentivirus will die within 72h 0m 0s while resistant survivors grow
4. Exchange the growth medium with puromycin-free medium to allow cell expansion; depending on the virus titre, allow 3 days for cells to grow
5. At confluency, lyse cells and test the knock-out efficiency by western blot using a mouse anti-target antibody, in this case, anti-GRK2 antibody

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Clone gRNA Vector

1. Digest Lenti-guide puro using Esp3I or BsmB1-V2 enzyme as follows: (Lenti-CRISPR-V2 can also be digested using the same enzyme)

A	B
Reagent:
Tango buffer 10X	5µl
Lentiguide puro vector (500ng/µl)	10µl
Esp3I or Bsmb1-V2	5µl
DTT 0.1M	2µl
Water	Up to 50µl

• Digest overnight at 37˚C
• A 2kb insert is generated by this cleavage; excise the upper band of the digested vector from the gel using a clean razor blade. Carry out standard DNA extraction from the agarose gel
• Dilute the cut vector to 20ng/µl and store at -20°C for further use

Prepare the Insert

• Order oligonucleotides to clone a guide RNA sequence using Addgene’s protocol on the lenti-guide puro webpage (https://media.addgene.org/data/plasmids/52/52963/52963-attachment_IPB7ZL_hJcbm.pdf). These oligos can alternatively be cloned into Lenti-CRISPR-v2
• Briefly, for each guide, two reverse complement oligos are ordered such that :

• Each oligo is diluted to 10µM and annealed and phosphorylated in a PCR tube as follows:

A	B
Reagent	Volume
10X T4 ligase buffer	2µl
Oligo 1 (10µM)	2µl
Oligo 2 (10µM)	2µl
T4 PNK	0.5µl
Water	13.5µl

• Transfer to a PCR machine

A	B
Reagent	Volume
Hold at 37°C	60 min
Ramp up to 95°C (fast mode)
95°C	3 min
Ramp down to 25°C	0.2°C/sec
25°C	10 min
4°C	hold

• Incubate in a PCR machine as follows:

A	B
Reagent	Volume
T4 ligase buffer 10x	1µl
Vector (20ng)	1µl
Insert (from previous step)	1µl
T7 ligase	1µl
0.1M ATP	0.5µl
0.1M DTT	0.5µl
Water	5µl

• Transform 5µl of the 10µl reaction into Dh5alpha competent cells
• Plate onto carbenicillin agar plates overnight
• To assess cloning efficiency, perform a colony PCR on the colonies 18 hr after transformation using the primers:

A	B
Forward	5’ ATCATATGCTTACCGTAACTTGAAAGTATTTCG
Reverse	5’ GACTCGGTGCCACTTTTTCAAG

• Pick a colony using a sterile tip and dilute into 10µl of Luria broth in a PCR tube
• Amplify a plasmid that has a guide cloned into the vector as a positive control
• Amplify a Bsmb1 digested plasmid as a negative control
• Amplify a colony from a control plate that didn’t receive an insert as a negative control
• Amplify the uncut plasmid as a negative control

These are the conditions for amplification of these plasmids:

A	B
Green GoTaq master mix 2X	12.5µl
Primer-Forward(10µM)	1µl
Primer-Reverse(10µM)	1µl
Bacterial suspension (diluted into 10µl)	1µl
Water	9.5µl

A	B	C
95°C	3 min
95°C	30 sec	Cycle 35 times
55°C	30sec
72°C	20 sec
72°C	1 min

• The green GoTaq PCR mix is ready to load onto a 2% agarose gel immediately after PCR
• The correct sized product is ~166bp
• Don’t forget to use a positive control (a cloned gRNA vector - 166bp band) and a negative control (uncut original lenti guide puro vector - 2kb band)
• Dilute the crude PCR product 1:10 and send for Sanger sequencing using the forward or reverse primer used for colony PCR
• Grow colonies that show the right sized band overnight at 37°C in 50µg/ml carbenicillin and perform a plasmid mini-prep
• Use the cloned plasmid for small scale lentivirus generation (dx.doi.org/10.17504/protocols.io.bp2l61z2zvqe/v1) if the Sanger sequencing shows the correct gRNA sequence has been cloned into the vector

Plate 1x105 3T3-Cas9 cells in one well of a 6 well plate containing 2ml complete DMEM medium (10% fetal bovine serum, penn/strep and Glutamax)* After 6 hours, add 0.5ml gRNA lentivirus plus 4µg/ml polybrene

• After 48 h, trypsinize and split the cells into 2 wells of a 6 well plate with 1 µg/ml puromycin
• After an additional 48-72 h, exchange the medium with complete DMEM without puromycin and let the cells grow for 2 more days
• At 80% confluence, trypsinize and plate 5x104 cells on coverslips in each well of a 24 well plate
• At the same time, take 20µl of the trypsinized cells and mix in 20µl QuickExtract solution to extract genomic DNA for knock out genotyping
• Perform QuickExtract genomic extraction for parental wild type 3T3 and lentivirus-gRNA infected cells
• Extract DNA according to this modified manufacturer’s protocol | A | B | | --- | --- | | 65°C | 6 min | | 98°C | 10 min | | 4°C | hold |

(Some protocols suggest 65°C for 10 min and that also works)

• PCR amplify a region around the gRNA-Cas9 cut site using genotyping PCR primers designed such that ~300-500bp are covered around the guide target site

For the gRNA used in the above example targeting mouse Rab12:

A	B
Forward	CCAAGGCCATGGTCATTCTTGATG
Reverse	TTACAACCCCAAACACTTGTTCCG

A	B
GoTax master mix 2X	12.5µl
Primer-Forward (10µM)	1µl
Primer-Reverse (10µM)	1µl
Quick Extracted genomic DNA	2µl
Water	8.5µl

A	B	C
95°C	2 min
95°C	30 sec	35 cycles
55°C	30 sec
72°C	45 sec
72°C	1 min

If the PCR band is of the correct size, dilute the sample 1:10 and Sanger sequence the crude PCR product using the reverse primer from the PCR

• Perform TIDE analysis (www.tide.nki.nl) or Synthego ICE analysis using the raw chromatograms in the .abi file provided with the sequencing results

• Percentage of cells with insertions or deletions (INDELs) is a proxy for knock-out efficiency

• The higher the percentage, the better the knock-out efficiency

• If needed, cells can be single cell sorted to create clonal knock-out lines for long term studies of the phenotype

• For 3T3 cells, increased passage number can be detrimental to cell physiology

• Best gRNAs with highest INDEL percentages are analyzed for the desired phenotype(s)

• Using the above mentioned gRNA directed against mouse Rab12, within 5 days we observed 80% efficiency

• Cells can be single cell sorted into 96 well plate using a Sony cell sorter at this point to create clonal cell lines with gene knock-outs