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CRISPR References and Information

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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.

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CRISPR Software

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

    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:

    1. 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
    2. 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 .

  • MAGeCK

    Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK). This tool can identify positively or negatively selected sgRNAs, genes, or pathways.

  • CrispyCrunch

    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.

gRNA Design

  • Deep learning models to design gRNAs and predict their efficiency.

  • GenET (Genome Editing Toolkit) (Link opens in a new window)is a library of various python functions for the purpose of designing and evaluating experiments.

  • 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.

  • CRISPOR

    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.

  • sgRNA Scorer 2.0
    From the Church Lab: a tool that identifies putative target sites for S. pyogenes Cas9, S. thermophilus Cas9, or Cpf from your input sequence or list of sequences.

  • 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.

  • CRISPRdirect
    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.

  • E-CRISP
    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

Protocols

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: pAC2, pAC152, pAC153, pAC154;
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 Cas9: pMLM3613
dsDNA donor plasmid backbones pSL1180-HR-PUbECFP & pSL1180polyUBdsRED 		PDF 583.3 KB
Zhang gRNA cloning CRISPR RNA array: Cas9 (pX260) or Cas9 D10A (pX334);
tracrRNA: Cas9 (pX330) or Cas9 D10A (pX335); 		PDF 248.6 KB
Zhang sgRNA cloning lentiCRISPR v2
packaging plasmids: pVSVg, psPAX2;
positive control: CMV-EGFP 		PDF 2.3 MB
Zhang GeCKO pooled library amplification 1 vector system: lentiCRISPR v2
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