CRISPR References and Information
In collaboration with the labs who have deposited CRISPR plasmids, we've created a series of links and guides to help you use CRISPR in your lab.
- Guide to CRISPR technology
- Introductory video from the Feng Zhang lab
- Addgene Blog Posts
- How to Design Your gRNA for CRISPR Genome Editing: John Doench from the Broad Institute gives practical advice for designing your gRNA and introducing it into cells.
- Protocol for Genomic Deletions in Mammalian Cell Lines
Sanger Indel Analysis
- ICE (Inference of CRISPR Edits)
This new open access tool from Synthego allows you to determine rates of CRISPR editing at a given locus. To use the tool, you'll need Sanger sequencing reads from PCR amplicons that cover your locus of interest and correspond to edited and non-edited cell populations. For more information, see the bioRxiv preprint describing ICE.
Deep Sequencing Data Analysis
CRISPResso is a robust and easy-to-use computational pipeline that enables accurate quantification and visualization of CRISPR-Cas9 outcomes, as well as comprehensive evaluation of their effect on the coding sequence, functional noncoding elements and off-target sites. This algorithm allows for the quantification of both non-homologous end joining (NHEJ) and homologous directed repair (HDR) frequency.
CRISPResso requires two inputs:
- paired-end reads (two files) or single-end reads (single file) in FASTQ format (fastq.gz files are also accepted) from a deep sequencing experiment
- a reference amplicon sequence to assess and quantify the efficiency of the targeted mutagenesis
The amplicon sequence expected after HDR can be provided as an optional input to assess HDR frequency. An sgRNA sequence (without PAM sequence) can be provided, to compare the predicted cleavage position to the position of the observed mutations. Coding sequence/s may be provided to quantify frameshift and potential splice site mutations.
The CRISPResso suite accommodates single or pooled amplicon deep sequencing, WGS datasets and allows the direct comparison of individual experiments. PubMed PMID 27404874 .
Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK). This tool can identify positively or negatively selected sgRNAs, genes, or pathways.
CrispyCrunch is used for high-throughput design and analysis of CRISPR + HDR experiments. For more information, see the Addgene blog post from the Chan Zuckerberg Biohub.
- Green Listed - a CRISPR Screen Tool
Design gRNAs for custom CRISPR screens targeting a long or short list of genes provided by the user. Read more about it on the Addgene blog.
- Eukaryotic Pathogen CRISPR guide RNA/DNA Design Tool (EuPaGDT)
gRNA design tool with extensive selection of eukaryotic pathogen genomes (200+) that can predict gRNA targets in gene families, HDR oligonucleotide design, and batch processing for designing genome-wide gRNA libraries. PubMed PMID 28348817.
This tool helps design (10 different prediction scores), clone (primer design), and evaluate gRNAs, as well as predict off-targets, for CRISPR in 180+ genomes. PubMed PMID: 27380939.
- RGEN (RNA-guided endonuclease) Tools: Cas-OFFinder & Cas-Designer
From the Kim Lab, Cas-OFFinder identifies gRNA target sequences from an input sequence and checks for off-target binding. Currently supports: Drosophila, Arabidopsis, zebrafish, C. elegans, mouse, human, rat, cow, dog, pig, Thale cress, rice (Oryza sativa), tomato, corn, monkey (macaca mulatta).
Cas-Designer searches for targets that maximize knockout efficiency while having a a low probability of off-target effects. Cas-Designer integrates information from the Kim Lab's Cas-OFFinder and Microhomology predictor.
- CRISPR-ERA: CRISPR-mediated editing, repression, and activation
From the Qi Lab, a sgRNA design tool for genome editing, as well as gene regulation (repression and activation). Genome support for bacteria (E. coli, B. subtilis), yeast (S. cerevisiae), worm (C. elegans), fruit fly, zebrafish, mouse, rat, and human.
- CCTop - CRISPR/Cas9 target online predictor
Identifies candidate sgRNA target sites by off-target quality. Validated for gene inactivation, NHEJ, and HDR. Reference genomes include Arabidopsis, C. elegans , sea squirt, cavefish, Chinese hamster, fruit fly, human, rice fish, mouse, silk worm, stickleback, tobacco, tomato, frog (X. laevis and X. tropicalis), and zebrafish.
- Off-Spotter: tool for CRISPR/Cas design
Program for designing optimal gRNAs. Provides feedback on number of potential off-targets, target's genomic location, and genome annotation. Available genomes are human (hg19 & hg38), mouse (mm10), and yeast (strain w303).
- CRISPR MultiTargeter
Can be used to identify novel gRNA target sites in a single gene, as well as a target site common to a set of similar sequences. Organisms include human, mouse, rat, chicken, frog, zebrafish, fly, worm, Japanese rice fish, maize, Arabidopsis, and rice. Proof-of-concept performed in zebrafish.
- ZiFiT Targeter
Originally developed to identify zinc finger nuclease sites, this tool from the Joung Lab has been expanded to identify potential DNA target sites for TALEs and CRISPR/Cas.
From the Database Center for Life Science (DBCLS) in Japan; Identify candidate gRNA target sequences in an input sequence, which can be an accession number, genomic location, pasted nucleotide sequence, or a sequence text file you upload. Currently supports: Human, mouse, rat, marmoset, pig, chicken, frog (X. tropicalis and X. laevis), zebrafish, sea squirt, Drosophila, C. elegans, Arabidopsis, rice, sorghum, silkworm, and budding and fission yeast.
- Target Finder (Feng Zhang lab)
Identifies gRNA target sequences from an input sequence and checks for off-target binding. Currently supports: Drosophila, Arabidopsis, zebrafish, C. elegans, mouse, human, rat, rabbit, pig, possum, chicken, dog, mosquito, and stickleback.
Identifies gRNA target sequences from an input sequence and checks for off-target binding. Currently supports: Drosophila, Arabidopsis, zebrafish, C. elegans , mouse, human, rat, yeast, frog, Brachypodium distachyon, Oryza sativa, Oryzias latipes
- CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes
From the Church Lab, a program that identifies gRNA target sequences from an input sequence, checks for off-target binding and can work for S. pyogenes, S. thermophilus or N. meningitidis Cas9 PAMs. Currently supports: mouse and human.
- CRISPR Optimal Target Finder
Tool from the O'Connor-Giles Lab that identifies gRNA target sequences from an input sequence and checks for off-target binding. Currently supports over 20 model and non-model invertebrate species.
Forums and FAQs
- Feng Zhang lab: Tips for CRISPR design, homology directed repair and cloning.
- CRISPR Discussion Group (moderated by Feng Zhang lab): Forum/FAQ
- Drosophila: General Advice and Forum/FAQ
- CRISPR Blog Topic Page: CRISPR tips, FAQs, and news featured on Addgene's blog
|Lab(s)||Description||Plasmids in protocol||Download protocol|
|Church||gRNA design and cloning||gRNA cloning vector||PDF (115.2 KB)|
|Church||gRNA design and cloning for Cas9 orthologs||Cas9 plasmids||PDF (104.1 KB)|
|Chen and Wente||Zebrafish: gRNA cloning, in vitro transcription, injection||gRNA core; Cas9; optimized Cas9||PDF (66.8 KB)|
|Fujii||gRNA design and cloning||
gRNA cloning vector
Retroviral vectors: neomycin (pSIR-neo), GFP (pSIR-GFP), DsRed (pSIR-DsRed-Express2), human CD2 (pSIR-hCD2)
|PDF (105.8 KB)|
|Fujii||iChIP/enChIP to purify genomic DNA||FLAG tagged dCas9||PDF (107.4 KB)|
|Goldstein||Nematode: gRNA design and cloning||Cas9-sgRNA construct||PDF (355.3 KB)|
|Goldstein||Nematode: Injection and selection for Cas9-triggered homologous recombination||Cas9-sgRNA target construct; pMA122 (negative selection marker); pGH8 (neuronal co-injection marker); pCFJ104 (body wall muscle co-injection marker); pCFJ90 (pharyngeal co-injection marker); pDD104 (Cre recombinase)||PDF (102.5 KB)|
|Jaenisch||CRISPR plasmid summary for transcriptional activation||
dCas9 activators sgRNA dual expression:
pmax dCas9 activator expression: pAC91, pAC92, pAC93, pAC94, pAC95;
dCas9 activator gateway donors: pAC84, pAC1, pAC147, pAC148, pAC149;
gateway destination: pAC90
|PDF (1.0 MB)|
|Katic||Nematode: Cas9 and gRNA use||Cas9 (pIK86); gRNA empty backbone (pDR274)||PDF (87.2 KB)|
|Liu||Fly: gRNA cloning||pAc-sgRNA-Cas9||PDF (165.1 KB)|
|Marraffini||Bacteria: pCas9 new spacer cloning||pCas9||PDF (125.6 KB)|
|Marraffini||Bacteria: pCRISPR new spacer cloning||pCRISPR||PDF (106.7 KB)|
|Mendenhall & Myers||Mammalian: FLAG tagging endogenous proteins||pFETCh_Donor; additional HDR & gRNA plasmids are available for CREB1, ATF1, GABPA, & RAD21||PDF (134.7 KB)|
|Musunuru||CRISPRs in human pluripotent stem cells||pCas9_GFP; gRNA empty vector||Link|
|O'Connor-Giles||Fly: gRNA and ssODNs design and cloning; injection protocol||pU6-BbsI-chiRNA; phsp70-Cas9||PDF (106.5 KB)|
|Orkin and Bauer||Protocol for Genomic Deletions in Mammalian Cell Lines||pSpCas9(BB) (pX330)||Link|
|Parrott||NEBuilder Assembly of CRISPR vectors using ssDNA oligos||p201G Cas9; p201B Cas9; p201H Cas9; p201N Cas9; pUC gRNA Shuttle||PDF (488.6 KB)|
|Sabatini and Lander||gRNA cloning into pLX-sgRNA||pLX-sgRNA||PDF (125.9 KB)|
|Sontheimer||gRNA design and cloning||All-in-one plasmid containing expression cassette for NmCas9 and BsmBI site for cloning in gRNA: pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s)||PDF (47.5 KB)|
|Vosshall and Matthews||CRISPR/Cas9 reagent generation; gRNA design; HDR design; Deep sequencing||
dsDNA donor plasmid backbones pSL1180-HR-PUbECFP & pSL1180polyUBdsRED
|PDF (583.3 KB)|
CRISPR RNA array:
Cas9 D10A (pX334);
tracrRNA: Cas9 (pX330) or Cas9 D10A (pX335);
|PDF (248.6 KB)|
packaging plasmids: pVSVg, psPAX2;
positive control: CMV-EGFP
|PDF (2.3 MB)|
|Zhang||GeCKO pooled library amplification||
1 vector system:
2 vector system: lentiCas9-Blast and lentiGuide-Puro
packaging plasmids: pVSVg, psPAX2
positive control: CMV-EGFP
Kits are also available (mouse or human libraries)
|PDF (269.2 KB)|