CRISPR/Cas Plasmids: 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 CRISPRs in your lab.
- Guide to CRISPR technologies
- Video from Dr. Feng Zhang's lab
- Addgene Blog Posts
How to Design Your gRNA for CRISPR Genome Editing
- John Doench and Ella Hartenian from the Broad Institute give practical advice for using CRISPRs: designing your gRNA and introducing it into cells.
- Validated gRNA Sequences : Search or deposit gRNA sequences that have been described in peer reviewed publications.
- Protocol for Genomic Deletions in Mammalian Cell Lines
Deep Sequencing Data Analysis
CRISPResso is as 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) occurrences. 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). Can identify positively or negatively selected sgRNAs, genes, or pathways.
Addgene's Validated gRNA Target Sequences DataTable
This table lists gRNA sequences that have been experimentally validated for use in CRISPR experiments. It is compiled from plasmids in Addgene's database as well as from sequences provided to us by users.
Quilt Universal guide RNA designer
Search for gRNAs via gene name or by genomic location. Database includes gRNAs from popular CRISPR libraries and from more than two million DNAse hypersensitive sites for intergenic guide RNAs in hg19, filtered for off-target effects.
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.
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 - for designing gRNAs
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).
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.
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.
Feng Zhang lab's Target Finder
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.
Michael Boutros lab's Target Finder (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
This software from the O'Connor-Giles Lab 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
|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 (212.7 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)|