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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