gRNA design tools and off-target prediction
Cloning guides and resources
CRISPR libraries
CRISPR analysis toolsgRNA design tools and off-target prediction:
First you have to design guide RNA (gRNA) recognition sites in you target sequence. The target has to be flanked by a PAM site (which is NGG in the case of the most widely used S. pyogenes Cas9). You want to design the gRNA as specific as you can, with limited homology to other regions in the genome. Please find below some online design tools that help you design the best gRNAs. Note however that the off-target predictions are very imprecise. You might want to run your gRNA on the CROP-IT, which is one of the best performing off-target identification software.
MIT, Feng Zhang lab CRISPR design tool
Available for gRNA design, nickase analysis and off-target prediction (for 15 different species). Gives the user a score based on the number of mismatches and the position of mismatches in the gRNA (PAM proximal binding is more stringent). Gives a quality assessment of each gRNAs based on homology to off-target sites.
E-CRISPR, Michael Boutros lab CRISPR design tool
Includes plenty of advanced design options. The user can select from 33 organisms for off-target search. Input is a user-defined target gene, either in FASTA format or gene symbol. User can define the purpose of application. Can search for NGG and NAG PAM sites as well. It is very useful that different regions of genes (5' part, 3' part, exons, avoids CpG islands etc.) can be specified in the input. Scores gRNAs based on efficacy, annotation and specificity.
ZiFiT, Keith Joung lab, genome editing design tool
This tool deigns CRISPR gRNAs, TALEN recognition sites and ZFNs. gRNAs has to be cloned into the MLM 3636 vector.
CasBLASTR, Keith Joung lab CRISPR design tool
This new tool allow to search for S. pyogenes, S. aureus and S. thermophylus Cas9 gRNA recognition sites. Furthermore it searches for NGA and NGCG PAM variant S. pyogenes Cas9 enzymes. Dimeric Cas9-FokI sites could also be searched for (spacer length could be defined by the user). It incorporates search for truncated gRNAs (tru-gRNAs) for enhanced specificity.
CROP-IT, CRISPR design and off-target analysis tool
The CRISPR design only allows 250bp sequences. The off-target analysis is very useful, it separates off-targets between cleavage and Cas9 binding (e.g. epigenome editing requires only binding of dCas9), since cleavage and binding have different tolerance to mismatches.
FlyCRISPR
A Drosophila focusing gRNA target searching algorithm.
ChopChop CRISPR design, Harvard
Allows for gRNA and TALEN recognition sites, recognition sequences are visualized along the gene of interest, off-target scoring. Incorporates some Cas9 variants. A useful feature is that it gives primer sequences for amplification.
CRISPRDirect, Database Center for Life Sciences, Japan
A simple CRISPR design tool, evaluates specificity of gRNAs.
Cloning guides and resources
Next, you have to clone your gRNA into one of the available backbones. Please find below some well-known Cas9 plasmids and respective cloning guides. Most of them are available through Addgene (click on the name to access it). A very useful guide is available through Addgene.
pX series, Feng Zhang plasmids
Available in different formats and selection markers:
pX330 - SpCas9 and single guide RNA
pX458 - SpCas9-2A-EGFP and single guide RNA (FACS sorting of transfected cells based on GFP)
pX459 - SpCas9-2A-Puro and single guide RNA (antibiotic selection of tranfected cells)
pX460 - SpCas9n (D10A nickase) and single guide RNA
pX461 - SpCas9n-2A-EGFP (D10A nickase) and single guide RNA
pX462 - SpCas9n-2A-Puro (D10A nickase) and single guide RNA
Cloning guides are available on the lab's website and in the Nature Protocols paper (available free in PMC).
Keith Joung plasmids
MLM3636 - gRNA cloning plasmid. Some cloning advice is found here.
JDS-246 - S. pyogenes Cas9 with 3xFLAG.
pSQT1313 - multiplex gRNA expression plasmid, should be used with chimeric dCas9-FokI, cloning guide is found here (look for the Supplementary Figure 8 in the paper).
pSQT1601 - dCas9-FokI expression plasmid, with Csy4 required to make gRNAs from pSQT1313
MLM3613 - Cas9 for zebrafish expression and DR274, gRNA expression for zebrafish
George Church plasmids
gRNA empty vector - synthesis and cloning guide is here.
hCas9 - human codon optimized Cas9
hCas9 D10A - nickase Cas9
CRISPR for transcriptional regulation
Activating proteins are usually based on dCas9-VP64 or BFP fusion proteins (d stands for dead, catalytically inactive Cas9). Inhibiting proteins are usually based on dCas9-KRAB fusion proteins.
dCas9-EGFP:
pSLQ1658 - Allows visualization of genomic loci with repetitive sequences.
CRISPR libraries
You can use a pool of different gRNAs to multiplex the CRISPR system. Here are some available libraries:
Jonathan Weissman libraries (CRISPRa and CRISPRi) for human cells
CRISPR is used to activate (CRISPRa) or inhibit (CRISPRi) gene expression. It can influence the transcription of 15,977 different genes. For CRISPRa, use the dCas9-VP64, for CRISPRi use the dCas9-KRAB.
Feng Zhang libraries:
SAM library is used for gene activation. Use with lenti-dCas9-VP64.
GecKO library is used for gene knockout. Available for human and mouse.
David Sabatini/Eric Lander libraries for human cells:
Lentivirus libraries are grouped into functional categories, including kinases, ribosomal proteins, cell cycle proteins, nuclear proteins. Use the control library for reference.
Kosuke Yusa mouse lentiviral library
CRISPR analysis tools:
CRISPResso - Next generation sequencing analysis tool. Requires FASTQ input files. It analyses NEHJ and HDR on the target locus.
