Check out our detailed JoVE protocol and accompanying video walkthrough for genome editing with CRISPR-Cas9 RNPs in primary human HSPCs and T cells, Caenorhabditis elegans, and Parhyale hawaiensis.
Additional protocols for an assortment of techniques related to CRISPR-Cas9 gene editing are available for free at protocols.io. Browse IGI-generated protocols, including:

• 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.

A variety of plasmids and pooled guide RNA libraries for CRISPR-mediated genome manipulation generated by IGI member labs and others can be acquired through Addgene. Our CRISPR screening facility also distributes certain gRNA libraries.

Published CRISPR interference, activation, or knockout cell lines may be provided upon request from the cell culture facility at UC Berkeley. Browse available lines here and contact Alison Killilea to learn more: ankillilea (at) berkeley.edu.

*You may wish to use a tool designed specifically for the organism you want to edit.*

“How to Make a Guide RNA for a Cas9 Knockout” tutorial (IGI)

CHOPCHOP (University of Bergen & Harvard Medical School)

CRISPR RGEN Suite (Institute for Basic Science, Korea; Seoul National University; Hanyang University)
Contains tools for identifying potential off-target sites (Cas-OFFinder) and microhomology regions (Microhomology-Predictor), as well as a guide RNA design tool that incorporates these parameters (Cas-Designer).

E-CRISP (DKFZ)

sgRNA Designer (CRISPRko & CRISPRa/i) (Broad)
Both tools work for S. pyogenes and S. aureus Cas9 (respective PAMs: NGG and NNGRR); use Doench cutting frequency determination (CFD) score to evaluate off-targets.

CRISPOR (Tefor)
Design gRNAs and accompanying primers for gRNA cloning, expression, and validating genomic edits. Displays multiple specificity scores, including Doench and Moreno-Mateos.

CRISPR-P (Huazhong Agricultural University)
Design gRNAs for genome editing in plants. Choose from PAMs corresponding to multiple Cas9/Cas12 variants and orthologs.

PrimeDesign (Harvard/MGH/Broad)
Design prime editing guide RNAs (pegRNAs) and nicking sgRNAs (ngRNAs) for prime editing experiments. Also includes PrimeVar, a database of candidate pegRNA/ngRNA pairs for correcting thousands of different pathogenic human mutations.

cld (CRISPR library designer) (Ontario Institute for Cancer Research)
Design multispecies sgRNA libraries.

CRISPOR Batch (Tefor)
Design gRNAs for lentiviral gene knockout screens in human or mouse cells.

CRISPRiaDesign scripts (UCSF)
Offers basic sgRNA machine learning scripts used to generate next-generation CRISPRi and CRISPRa libraries from Jonathan Weissman’s lab.

A primer on CRISPRi/a cell line production (UCSF)
The Weissman lab's handy guide on how to generate and validate stably-expressing CRISPRi and CRISPRa cell lines.

Pooled Sanger sequencing data

TIDE & TIDER (Desktop Genetics; Netherlands Cancer Institute)
Analyze Sanger sequencing traces from pools of cells to quantify indel spectrum around cut site (TIDE) or templated mutations (TIDER).

NGS data

CasAnalyzer (Institute for Basic Science, Korea; Seoul National University; Hanyang University)
Offers JavaScript-based instant assessment of genome editing NGS data.

CRISPResso (Mass General Hospital & Harvard)
Analyze deep sequencing data to determine outcomes after editing genomes with Cas9, Cas12, base editors, prime editors, etc.

CRISPR screen data

caRpools (Ontario Institute for Cancer Research)
Analyze raw NGS data from pooled CRISPR screens.

MAGeCK (Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout) (Dana-Farber & Harvard)
Analyze data from genome-wide CRISPR knockout screens.

ScreenProcessing (UCSF)
Analyze sequencing data from pooled CRISPR screens.

CRISPRAnalyzeR (DKFZ)
User-friendly, web-based analysis suite for pooled CRISPR-Cas9 screens.

Third Wave Analytics
The IGI Clinical Laboratory has relied on the LIMS from Third Wave Analytics since the launch of our COVID testing in 2020, and continues to use it for other applications in in the Interventional Genomics Unit.

CRISPRCasdb, CRISPRCasFinder, and other CRISPR-Cas++ tools (University of Paris-Saclay)
Find cas genes CRISPR arrays in a prokaryotic species of interest from a database of analyzed genomes (db) or in a user-uploaded sequence (Finder).

Bondy-Denomy Lab's List of Anti-CRISPRs (UCSF)
This sheet includes all published anti-CRISPRs, including their name, amino acid sequence, the CRISPR system(s) they inhibit, and other details.

GenomeCRISPR (DKFZ)
This database compiles results from CRISPR–Cas9 high-throughput screening studies, featuring data from over half a million guide RNAs in 421 human cell lines.

ClinVar (NIH)
A searchable database of human genomic variants and known health consequences.

ClinicalTrials.gov (NIH)
A searchable database of clinical studies conducted around the world. For example, enter "CRISPR" in the "Other terms" field and then click "Search" to see all current clinical trials that involve CRISPR technologies.

Addgene’s List of CRISPR Resources