CRISPR gRNAs cloning 

To design your gRNAs, use CRISPick portal

(https://portals.broadinstitute.org/gppx/crispick/public).

Order the Oligos with specific overhangs for BsmBI cloning.

Insert the designed 20bp target gRNA sequence between the overhangs.

Forward oligo: 5’ CACCG.......20 bp target.........-3’

Reverse oligo: 5’ AAAC......20 bp.........C 3’

Preparation of the gRNA oligonucleotides.

Spin oligonucleotide tubes briefly.

Dilute to 100micromolar (µM) solution with water.

Vortex, leave for some minutes, and vortex again.

Annealing of oligonucleotides:

• 100micromolar (µM) of oligo A (forward)

• 100micromolar (µM) of oligo B (reverse)

• 2µL 10x NEB buffer 2

• water up to 20µL total reaction volume

Denature at 95°C for 0h 5m 0s, then cool down slowly.

When they have reached Room temperature, spin down.

Prepare the assembly reaction for each oligonucleotide in individual PCR tubes containing:

1. 100ng backbone of lentiviral plasmid of choice (make sure it includes the AmpR gene for selection)
2. 1µL of annealed gRNA oligonucleotide from step 1
3. 1µL BsmBI/Esp3I restriction enzyme. FAST DIGEST
4. 1µL T4 DNA ligase
5. 2µL 10x T4 ligase buffer (to a final concentration of 1x)
6. Nuclease-free water up to20µL total reaction volume

Incubate the reaction in a thermal cycler with the following conditions:

• 10 cycles 0h 5m 0s at 37°C.

                   `0h 10m 0s` at  `22°C`. 
  

• Hold for 0h 30m 0s at 37°C .

• Hold for 0h 15m 0s at 75°C.

• Keep at 4°C.

Thaw Stbl3 or homemade top10 competent bacteria 37On ice.

Add 2µL of the ligation reaction from step 2 to the bacteria 37On ice.

Mix a little by tapping the tube carefully a couple of times.

Keep 37On ice for 0h 30m 0s.

To transform, dip the tubes in a 42°C water bath for exactly 0h 0m 45s.

Put the tubes back 42On ice for 0h 2m 0s.

Transfer the bacteria to 250µL pre-warmed or 42Room temperature soc media in a ventilated 15 ml falcon tube.

Incubate at 37°C with shake for 1h 0m 0s.

Spread everything on pre-warmed ampicillin+ agar plates.

Incubate at 37°C 1h 0m 0s.

The day after, pick up 3 different colonies for each of the plasmids from the agar plates and prepare 3 minipreps. Incubate the minipreps 1h 0m 0s on shake at 37°C.

Isolate the plasmid from the minipreps using the GeneJet Plasmid Miniprep kit (ThermoFisher).

Measure the DNA concentration.

Digest the DNA using the AfIII (BspTI) restriction enzyme to linearize the plasmid.

Check for the correct plasmid size on 1% agarose gel.

If the plasmids have the correct size, send for sequencing 1 or 2 for each cloned gRNA..

If the sequencing confirms the correct plasmid sequence, use one of the sequenced miniprep for each gRNA to prepare a maxiprep. Incubate the maxipreps 1h 0m 0s at 37°C.

Isolate the plasmid from the maxipreps using the NucleoBond Xtra Midi Plus Ef (ThermoFisher).

Measure the DNA concentration.

Digest the DNA using the AfIII (BspTI) restriction enzyme to linearize the plasmid.

Check for correct plasmid size on 1% agarose gel, and send for sequencing.