TITLE: CRISPR gRNAs cloning 
AUTHORS: Anita Adami

[DESCRIPTION]
This protocol details the procedure of CRISPR gRNAs cloning.

[STEPS]
SECTION: gRNA oligonucleotides design
1. To design your gRNAs, use CRISPick portal

 (https://portals.broadinstitute.org/gppx/crispick/public).
SECTION: gRNA oligonucleotides cloning
2. Preparation of the gRNA oligonucleotides.
SECTION: gRNA oligonucleotides cloning
2.1. Spin oligonucleotide tubes briefly.
SECTION: gRNA oligonucleotides cloning
3. Prepare the assembly reaction for each oligonucleotide in individual PCR tubes containing:
 100 ng backbone of lentiviral plasmid of choice (make sure it includes the AmpR gene for selection)
 1 µL  of annealed gRNA oligonucleotide from step 1
 1 µL BsmBI/Esp3I restriction enzyme. FAST DIGEST
 1 µL T4 DNA ligase
 2 µL 10x T4 ligase buffer (to a final concentration of 1x)
Nuclease-free water up to20 µL total reaction volume
SECTION: gRNA oligonucleotides cloning
2.3. Vortex, leave for some minutes, and vortex again.
SECTION: gRNA oligonucleotides cloning
2.2. Dilute to 100 micromolar (µM) solution with water.

SECTION: gRNA oligonucleotides cloning
2.4. Annealing of oligonucleotides: 

100 micromolar (µM)  of oligo A (forward) 

100 micromolar (µM)  of oligo B (reverse) 

2 µL 10x NEB buffer 2 

water up to 20 µL  total reaction volume

SECTION: gRNA oligonucleotides cloning
2.5. Denature at 95 °C for 5 min, then cool down slowly.


 

SECTION: gRNA oligonucleotides cloning
2.6. When they have reached Room temperature, spin down.

SECTION: gRNA oligonucleotides cloning
3.1. Incubate the reaction in a thermal cycler with the following conditions:

10 cycles 5 min at  37 °C. 

                       10 min at  22 °C. 

Hold for 30 min at  37 °C .

Hold for 15 min at  75 °C.

 Keep at  4 °C.

SECTION: Plasmid transformation &amp; preparation
4. Thaw Stbl3 or homemade top10 competent bacteria on ice.

SECTION: Plasmid transformation &amp; preparation
4.1. Add 2 µL of the ligation reaction from step 2 to the bacteria on ice.

SECTION: Plasmid transformation &amp; preparation
4.2. Mix a little by tapping the tube carefully a couple of times.
SECTION: Plasmid transformation &amp; preparation
4.3. Keep on ice for  30 min.

SECTION: Plasmid transformation &amp; preparation
4.4. To transform, dip the tubes in a 42 °C water bath for exactly 45 s.

SECTION: Plasmid transformation &amp; preparation
4.6. Transfer the bacteria to 250 µL pre-warmed or 42 Room temperature soc media in a ventilated 15 ml falcon tube.

SECTION: Plasmid transformation &amp; preparation
4.8. Spread everything on pre-warmed ampicillin+ agar plates.


SECTION: Plasmid transformation &amp; preparation
5. The day after, pick up 3 different colonies for each of the plasmids from the agar plates and prepare 3 minipreps. Incubate the minipreps 60 min on shake at 37 °C.
SECTION: Plasmid transformation &amp; preparation
6. Isolate the plasmid from the minipreps using the GeneJet Plasmid Miniprep kit (ThermoFisher).
SECTION: Plasmid transformation &amp; preparation
6.1. Measure the DNA concentration.
SECTION: Plasmid transformation &amp; preparation
6.2. Digest the DNA using the AfIII (BspTI) restriction enzyme to linearize the plasmid.

 

SECTION: Plasmid transformation &amp; preparation
6.3. Check for the correct plasmid size on 1% agarose gel.
SECTION: Plasmid transformation &amp; preparation
6.4. If the plasmids have the correct size, send for sequencing 1 or 2 for each cloned gRNA..
SECTION: Plasmid transformation &amp; preparation
7.  If the sequencing confirms the correct plasmid sequence, use one of the sequenced miniprep for each gRNA to prepare a maxiprep. Incubate the maxipreps  60 min at 37 °C.
SECTION: Plasmid transformation &amp; preparation
4.5. Put the tubes back on ice for 2 min.

SECTION: Plasmid transformation &amp; preparation
4.7. Incubate at 37 °C with shake for 60 min.

SECTION: Plasmid transformation &amp; preparation
4.9. 
Incubate at 37 °C  60 min.

 

SECTION: gRNA oligonucleotides design
1.1. Order the Oligos with specific overhangs for BsmBI cloning. 
SECTION: gRNA oligonucleotides design
1.2. 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’
SECTION: Plasmid transformation &amp; preparation
8. Isolate the plasmid from the maxipreps using the NucleoBond Xtra Midi Plus Ef (ThermoFisher).
SECTION: Plasmid transformation &amp; preparation
8.1. Measure the DNA concentration.
SECTION: Plasmid transformation &amp; preparation
8.2. Digest the DNA using the AfIII (BspTI)  restriction enzyme to linearize the plasmid. 
SECTION: Plasmid transformation &amp; preparation
8.3. Check for correct plasmid size on 1% agarose gel, and send for sequencing.