- Cas9 protein expression and purification
- In vitro transcription of guide RNAs
- Cas9 RNP nucleofection
- Genomic DNA extraction and PCR
- Re-amplification of CRISPRi and CRISPRa libraries
Protocols used by our CRISPR screening facility can be found here, including:
- Cloning, sequencing, and re-amplifying gRNA libraries
- Generating stable cell lines expressing Cas9
- Large-scale lentivirus production and transfection
- Screening adherent and suspension cells
Larger collections of protocols are also available:
CRISPR-Cas: A laboratory manual. Edited by Doudna JA and Mali P. Cold Spring Harbor Laboratory Press (2016).
Contains a variety of protocols for genome manipulation with Cas9, including genome editing, troubleshooting in specific organisms, how to detect editing, guide RNA library construction, and CRISPR inhibition or activation screening.
CRISPR Methods and Protocols. Edited by Lundgren M, Charpentier E, and Fineran PC. Methods in Molecular Biology (2015).
Features protocols for studying the mechanisms of CRISPR-Cas immunity from genetic, genomic, molecular, biochemical, and structural perspectives.
Genome Editing in Animals. Edited by Hatada I. Methods in Molecular Biology (2017).
Covers methods for editing genomes of many different animals, including insects, fish, and mammals.