Sep 17, 2025
CRISPR knockout protocol
- Nicholas Pease1
- 1University of Pittsburgh
Protocol Citation: Nicholas Pease 2025. CRISPR knockout protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmbpr5g3p/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: September 16, 2025
Last Modified: September 17, 2025
Protocol Integer ID: 227460
Keywords: crispr knockout protocol, crispr knockout, sequencing, protocol, cell rna
Abstract
This is a protocol used to perform CRISPR knockouts in primary human B cells to be subsequently profiled by single-cell RNA-sequencing. It has been adapted from Synthego and Lonza user manuals.
Materials
- sgRNAs (20uM stock, Synthego)
- P3 nucleofector solution (Lonza)
- Cas9
- PBS
- Nucleocuvette strips (Lonza)
- 24 well flat-bottom tissue culture plates
Troubleshooting
CRISPR KO protocol
Thaw sgRNAs (20uM stock, Synthego) at RT for 15-30 minutes.
Centrifuge sgRNA stocks for 5min at 1000xg.
Pre-warm culture media at 37C.
Add supplement 1 to P3 nucleofector solution (Lonza) at 4.5:1 ratio.
Assemble ribonucleoprotein (RNP) complexes by adding 60 pmol of sgRNA to 20 pmol of Cas9 diluted in supplemented P3 nucleofector solution (total volume of 20uL). Incubate for 10-60 minutes at room temperature.
Centrifuge 300,000 cells per nucleofection at 90xg for 10 minutes.
Remove supernatant and resuspend in 5mL of PBS and centrifuge at 90xg for 10 minutes.
Remove supernatant and resuspend cells in 5uL of supplemented P3 nucleofector solution per nucleofection.
Add 5uL of diluted cells to each RNP complex (total volume of 25uL).
Mix cell-RNP mixture and transfer to Nucleocuvette strips (Lonza). Nucleofect cells with CA-137 pulse code.
Add 75uL of pre-warmed culture media, mix and transfer cells to 24 well flat-bottom tissue culture plates containing 400uL of pre-warmed culture media.