Mar 15, 2024
CRISPR gRNAs cloning
- 1Laboratory of Molecular Neurogenetics, Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, BMC A11, Lund University, 221 84 Lund, Sweden.
Protocol Citation: Anita Adami 2024. CRISPR gRNAs cloning . protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9yj3pg3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 23, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 57294
Keywords: CRISPR gRNAs cloning, gRNA oligonucleotides, ASAPCRN, procedure of crispr grnas cloning, crispr grnas cloning, crispr, procedure, crispr grna
Funders Acknowledgements:
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-000520
Swedish Research Council
Grant ID: 2018-02694
Swedish Brain Foundation
Grant ID: FO2019-0098
Cancerfonden
Grant ID: 190326
Barncancerfonden
Grant ID: PR2017-0053
NIHR Cambridge Biomedical Research Centre
Grant ID: NIHR203312
Swedish Society for Medical Research
Grant ID: S19-0100
National Institutes of Health
Grant ID: HG002385
Swedish Research Council
Grant ID: 2021-03494
Swedish Research Council
Grant ID: 2020-01660
Abstract
This protocol details the procedure of CRISPR gRNAs cloning.
Attachments
gfccbg5jf.pdf
40KB
gRNA oligonucleotides design
To design your gRNAs, use CRISPick portal
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’
gRNA oligonucleotides cloning
Preparation of the gRNA oligonucleotides.
Spin oligonucleotide tubes briefly.
Dilute to 100 micromolar (µM) solution with water.
Vortex, leave for some minutes, and vortex again.
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
Denature at 95 °C for 00:05:00 , then cool down slowly.
Note
Recommended: Turn the heating block of and leave the tubes in it for 02:00:00 - 03:00:00
5m
When they have reached Room temperature , spin down.
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
Incubate the reaction in a thermal cycler with the following conditions:
- 10 cycles 00:05:00 at 37 °C .
00:10:00 at 22 °C .
- Hold for 00:30:00 at 37 °C .
- Hold for 00:15:00 at 75 °C .
- Keep at 4 °C .
1h
Plasmid transformation & preparation
4h 32m 45s
Thaw Stbl3 or homemade top10 competent bacteria On ice .
Add 2 µL of the ligation reaction from step 2 to the bacteria On ice .
Mix a little by tapping the tube carefully a couple of times.
Keep On ice for 00:30:00 .
30m
To transform, dip the tubes in a 42 °C water bath for exactly 00:00:45 .
45s
Put the tubes back On ice for 00:02:00 .
2m
Transfer the bacteria to 250 µL pre-warmed or Room temperature soc media in a ventilated 15 ml falcon tube.
Incubate at 37 °C with shake for 01:00:00 .
1h
Spread everything on pre-warmed ampicillin+ agar plates.
Incubate at 37 °C Overnight .
Note
Only successfully transformed colonies will grow on the plate.
1h
The day after, pick up 3 different colonies for each of the plasmids from the agar plates and prepare 3 minipreps. Incubate the minipreps Overnight on shake at 37 °C .
1h
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.
Note
This restriction site is part of the LTR found in lentiviral plasmids.
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 Overnight at 37 °C .
1h
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.