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Table 2 All-in-one packages for the design of guide RNAs and prediction of off-target effects

From: Resources for the design of CRISPR gene editing experiments

Tool Website Reference Input Output Throughput a Use cases Genomes sgRNA sequence constraints b Validation Pros Cons Software
CCTop http://crispr.cos.uni-heidelberg.de/ [68] Sequences 23 to 500 bp (similar to crispr.mit input); also has single or batch mode Scores OTs and ranks sgRNA by OTs. OT sites; position with respect to CDS. Number and position of MMs Low Find target sequences for a sequence or sequences (has batch mode); good for generating a lot of candidates with comprehensive on/off-target information Approximately 15 common organisms; only a single human build available NGG or NRG PAMs for on- and off-target. 5′ G or GGa. MM tolerance, total and in core. Annotated OT sites with RefSeq IDs, if applicable On- and off-target efficacy in vitro. For one guide Ease of use, many options. Comprehensive and easy-to-understand output No on-target efficacy prediction. Does not account for identity of MMs. Regular mode is relatively fast. Advanced mode is slow Web
CHOPCHOP https://chopchop.rc.fas.harvard.edu/ [49] Target transcript ID or sequence (raw or chromosomal position); also allows gene input Scores OT and ranks sgRNA by off-targets, GC content, genomic map, position with respect to CDS, primers for validation, RE sites. Off-target sites 0–2 MM Low/medium Find target sequences for a single sequence/gene/transcript; good for generating a lot of guides for a single target quickly Approximately 20 common organisms, plus two most recent builds for human 5′ GN, GG no TTTT, CDS, junctions, alternative PAMs; specify restrictions for position of mismatch, e.g., nine-nucleotide 5′ to PAM (seed)   Ease of use, flexible options, downloadable results No on-target efficacy prediction. OT limited to zero to two mismatches. Does not account for identity of mismatches Web
Crispr.mit http://crispr.mit.edu/ [65] Sequence or FASTA files; single or batch mode Scores OT and ranks sgRNA by OTs, paired sgRNAs for nickase, OT sites Low/medium Find target sequences for a sequence or sequences (has batch mode); good for generating a lot of candidates with comprehensive on/off-target information Approximately 15 common organisms; only a single human build available Paired sgRNAs   Ease of use. Scores OTs for up to four MMs and provides positions. Specifies OTs in genes versus intergenic sequences Handles short sequences 23–500 bp, although 250 bp is actual upper limit. Very slow. No efficacy metric. Does not account for identity of mismatches. Occasionally misses OT sites with no MMs Web
WU-CRISPR http://crispr.wustl.edu [104] Gene symbol or 24–30,000 bp sequence List of sgRNA ranked by efficacy score Low Find target seqs based on efficacy score and absence of OT perfect seed match Mouse and human None   Ease of use. On-target efficacy scores based on re-analysis from [37] OT exclusion: perfect 13-nt seed match or >85 % similarity of 20-nt sequence to exome. Doesn’t account for identity of mismatches web
GT-Scan http://gt-scan.braembl.org.au/gt-scan/ [69] Sequence or FASTA file. Genomic coordinates sgRNA, genomic sites with zero to three mismatches. Links to genome browser Low Find target sequences and OTs for a single sequence Approximately 20 common organisms; only a single human build available Many user-defined OT rules and filters. Alternative PAMs   Ease of use. Many filters to define OT rules Has trouble finding exact matches in genome. Does not account for identity of mismatches Web
CROP-IT http://www.adlilab.org/CROP-IT/homepage.html [58] sgRNA Lists OT sites. Scoring for OT sites. Number, position, identity of MMs. Genomic position, CDS gene name if relevant. Email results Low Find off-target information for a guide sequence Mouse and human Cas9 binding sites versus predicted cleavage sites. NGG or NNG PAMs   Ease of use. Provides gene name if OT is in exon Analyzes one sgRNA at a time. Does not account for identity of mismatches.
Email response slow
Web
Cas OT http://eendb.zfgenetics.org/casot/ [44] FASTA files sgRNA and OT sites Low/medium Target sequences and OTs Provided by user Several OT rules and restrictions   Many options. Alternative PAMs, paired sgRNAs, 5′ G Programming knowledge necessary or helpful. Does not account for identity of mismatches Perl script
CRISPRseek http://www.bioconductor.org/packages/release/bioc/html/CRISPRseek.html [48] Software package Candidate sgRNAs with a variety of scores, dependent on parameters High Find target sequences and OT sites for multiple sequences; performs both nickase and paired guide design Several common genomes Many options. Very comprehensive in terms of design and scoring   Many options Very laborious to both install and operate, despite having extensive documentation. Does not account for identity of mismatches Bioconductor package in R
ZiFiT http://zifit.partners.org/ZiFiT/ [105, 106] Input sequence sgRNA, OTs with zero to three MMs. Position and identity of MM. Genomic position of OT Low Find target sequences and OTs for a single sequence Nine common genomes 5' G or GG   Ease of use No on-target efficacy prediction. Does not account for identity of mismatches Web
E-CRISP http://www.e-crisp.org/E-CRISP/ [47] Gene symbol/sequence Many options for output in advanced mode; table provides sequence, three-part score (specificity, annotation of gene target regions hit, and on-target efficacy), context, number of hits Low Find target sequences for a single gene or sequence; performs guide evaluation as well (not tested); also cas9 nickase design for paired sgRNAs >30 genomes Basic mode: user defines MM tolerance, PAM sequence. Advanced mode: many options for OT specs   Lots of options, fast results, summary of all designs found So many options — could be confusing. Does not account for identity of mismatches Web
CRISPR Direct http://crispr.dbcls.jp/ [107] Transcript/genome location/nucleotide sequence Table with target position, sequence plus context, some sequence info (GC content, Tm, poly-T), target counts plus downloads Low Find target sequences for a single transcript/sequence with some limited off-target info 20 common genomes PAM type   Very fast, visual display of target sequence and OT info No options, no on-target efficacy, OT matches limited to number of target sites with 20/12/8-mer plus PAM matches. Does not account for identity of mismatches Web
COD http://cas9.wicp.net N/A 23–400 bp input sequence GenBank file and CSV file. OT scoring. Position, identity, number of MMs. Genomic position of OT site with link to graphic Low Find target sequences for an input sequence. Predicts and ranks OT sites 23 common genomes Length of target. OT stringency. NGG and/or NAG for OT   Ease of use. OT scoring No on-target prediction. Does not account for identity of mismatches. Slow Web
sgRNAcas9 http://www.biootools.com/col.jsp?id=103 [45] Software package Multiple files that include all possible designs for a given sequence plus information to filter based on cloning or on-target efficacy, OT information High Find target sequences for a sequence or sequences (has batch mode); good for generating a lot of candidates, with some limited on-target or OT information User can provide any genome reference file Can generate single or paired sgRNAs; many options for OT stringency (number of mismatches and number of offsets); several options for ease of cloning   Can generate several candidates, with some (but not a comprehensive set of) options with respect to cloning ease and efficiency and OT matching Difficult to use. No clear on-target efficacy score. Supports only NGG PAM currently. Does not account for identity of mismatches Local (Perl script)
DNA 2.0 gRNA Design Tool https://www.dna20.com/eCommerce/cas9/input N/A Gene, locus, sequence Display of targets; table: hit position, target sequence in context, score, overlapping gene info, number of splice variant targets Low Find target sequences for a single gene or sequence; performs cas9 and nickase design Human, mouse, Escherichia coli, Arabidopsis, yeast PAM type   Simple interface, very fast results, simple and clear output Tied in to commercial site; output has very few data points, and unclear what is available with respect to scoring. Does not account for identity of mismatches Web/commercial
Cas-Designer http://www.rgenome.net/cas-designer/ N/A Nucleotide sequence or FASTA Target sequence and OT. Zero to two MMs and one bulge. Percentage GC. Link to Ensembl for OT sites Low Find target sequences. Uses Cas-OFFinder and Microhomology Predictor for OT searching 30 genome builds Accounts for bulge MMs. Alternative PAMs   Ease of use. Fast. Accommodates bulges in OT prediction No on-target efficacy score. OT prediction does not account for identity Web
sgRNA Scorer 1.0 https://crispr.med.harvard.edu/sgRNAScorer/
Input
[62] Nucleotide sequence. FASTA files up to 10 kb sequences Target sequence with activity score. Number of OT sites with genomic coordinates Low Find target sequences OT searching using CasFinder. On-target activity scoring using support vector machine (SVM) model 12 common genomes Streptococcus pyogenes or S. thermophiles PAMs   On-target efficacy score. Offers a precomputed list of target sequences for all human and mouse genes Slow. Email output. OT prediction does not account for identity Web
Protospacer http://www.protospacer.com/ [51] Many inputs. Gene ID, genomic coordinates, etc. Target sequence with activity score. Percentage GC, OT sites, positions, identities Medium/high Find target sequences, OT sites, prioritize sgRNAs Provided by user Accounts for MMs. Alternative PAMs   On-target efficacy score based on sgRNA Designer rules (Table 1). Many options for assessing OT Requires extensive setup. Not obvious where OTs fall with respect to CDS. OT prediction does not account for identity Software,
local
  1. a‘Low’: input format and run times support one-gene-at-a-time or one-guide-at-a-time queries. ‘Medium’: input format and run times support small batches of genes or sgRNAs, tens to hundreds of queries. ‘High’: input format and run times support genome-scale queries. bOptions for sgRNA sequence criteria: alternative PAMs. Require 5′ G to promote PolIII-dependent transcription from the U6 promoter, or 5′ GG for in vitro transcription using the T7 polymerase. Avoid TTTT, which signals PolIII transcriptional termination.
  2. Abbreviations: CDS coding sequence, MM mismatch, N/A not applicable, OT off-target, PAM protospacer adjacent motif, sgRNA single guide RNA, Tm melting temperature