Mar 15, 2024

Public workspaceCRISPR gRNAs cloning 

DOI
dx.doi.org/10.17504/protocols.io.x54v9yj3pg3e/v1
  • 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.
Danielle Daft
Open access
DOI: dx.doi.org/10.17504/protocols.io.x54v9yj3pg3e/v1
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
Funders Acknowledgement:
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
gfccbg5jf.pdf
40KB
gRNA oligonucleotides design
gRNA oligonucleotides design
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’
gRNA oligonucleotides cloning
gRNA oligonucleotides cloning
Preparation of the gRNA oligonucleotides.
Spin oligonucleotide tubes briefly.
Centrifigation
Dilute to Concentration100 micromolar (µM) solution with water.

Vortex, leave for some minutes, and vortex again.
Mix
Annealing of oligonucleotides:

  • Concentration100 micromolar (µM) of oligo A (forward)

  • Concentration100 micromolar (µM) of oligo B (reverse)

  • Amount2 µL 10x NEB buffer 2

  • water up to Amount20 µL total reaction volume

Pipetting
Denature at Temperature95 °C for Duration00:05:00 , then cool down slowly.


Note
Recommended: Turn the heating block of and leave the tubes in it for Duration02:00:00 - Duration03:00:00

5m
When they have reached TemperatureRoom temperature , spin down.

Centrifigation
Prepare the assembly reaction for each oligonucleotide in individual PCR tubes containing:
  1. Amount100 ng backbone of lentiviral plasmid of choice (make sure it includes the AmpR gene for selection)
  2. Amount1 µL of annealed gRNA oligonucleotide from step 1
  3. Amount1 µL BsmBI/Esp3I restriction enzyme. FAST DIGEST
  4. Amount1 µL T4 DNA ligase
  5. Amount2 µL 10x T4 ligase buffer (to a final concentration of 1x)
  6. Nuclease-free water up toAmount20 µL total reaction volume
Pipetting
Incubate the reaction in a thermal cycler with the following conditions:

  • 10 cycles Duration00:05:00 at Temperature37 °C .

Duration00:10:00 at Temperature22 °C .

  • Hold for Duration00:30:00 at Temperature37 °C .

  • Hold for Duration00:15:00 at Temperature75 °C .

  • Keep at Temperature4 °C .

1h
Incubation
PCR
Plasmid transformation & preparation
Plasmid transformation & preparation
4h 32m 45s
Thaw Stbl3 or homemade top10 competent bacteria TemperatureOn ice .

Add Amount2 µL of the ligation reaction from step 2 to the bacteria TemperatureOn ice .

Pipetting
Mix a little by tapping the tube carefully a couple of times.
Mix
Keep TemperatureOn ice for Duration00:30:00 .

30m
To transform, dip the tubes in a Temperature42 °C water bath for exactly Duration00:00:45 .

45s
Put the tubes back TemperatureOn ice for Duration00:02:00 .

2m
Transfer the bacteria to Amount250 µL pre-warmed or TemperatureRoom temperature soc media in a ventilated 15 ml falcon tube.

Incubate at Temperature37 °C with shake for Duration01:00:00 .

1h
Incubation
Spread everything on pre-warmed ampicillin+ agar plates.



Incubate at Temperature37 °C DurationOvernight .

Note
Only successfully transformed colonies will grow on the plate.

1h
Incubation
Overnight
The day after, pick up 3 different colonies for each of the plasmids from the agar plates and prepare 3 minipreps. Incubate the minipreps DurationOvernight on shake at Temperature37 °C .
1h
Incubation
Overnight
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.

Digestion
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 DurationOvernight at Temperature37 °C .
1h
Incubation
Overnight
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.
Digestion
Check for correct plasmid size on 1% agarose gel, and send for sequencing.