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Table 4 Tools available for sgRNA designing

From: CRISPR/Cas9: the Jedi against the dark empire of diseases

Tool Website Purpose Input Output Available genomes Pros Cons Reference
Atum https://www.atum.bio/eCommerce/cas9/input Candidate gRNA DNA sequence, (max 10,000 bp) gene name, genomic region Candidate guidesequences and off target loci 5 Uses ATUM scoring to minimize off-targets/all in one nickase ninja Not free of cost N/A
Benchling https://benchling.com/crispr Candidate gRNA DNA sequence/gene name Candidate guidesequences and off target loci 5 Free of cost   N/A
CRISPR design http://crispr.mit.edu/ Used to find target sequences for a sequence or seuqnces (batch mode), also provides in depth on/off target information DNA sequence/FASTA files single or batch mode Candidate guide sequences and off target loci 16 Helpful to generate many candidates with on/off information Only handles short sequences up to23–500 bp/slow to use/no efficacy merit/does not indicate identity of mismatches [39]
CHOPCHOP https://chopchop.rc.fas.harvard.edu/ To find target sequences for a single sequence/gene/transcript DNA sequence/gene name/genome location Candidate guide sequences and off target loci, 23 Good to generate multiple guides for a single target, free of cost No on-target efficacy [40]
Cas off finder http://www.rgenome.net/cas-offinder/ Provides information about OTs without limiting the PAM or number of mismatch bases sgRNA Off target loci for guide sequences 20 Free of cost/easy to use Does not give indication if OTs are in coding sequences [41]
Fly CRISPR http://tools.flycrispr.molbio.wisc.edu/targetFinder/ Candidiate gRNA, software provides maximum stringency-(uses strict algorithm based on OT cleavage in cells lines) and minimum stringency; off target cleavage effects observed DNA sequence Candidate guidesequences and off target loci, 18 Free of cost Slow to use [42]
E-CRISP http://www.e-crisp.org/E-CRISP/designcrispr.html Finds target sequences for a single gene or sequence DNA sequence/gene symbol Candidate guide sequences and off target loci, 30 Free of cost Numerous options maybe confusing/no account for identity of mismatches [43]
Cas OT http://eendb.zfgenetics.org/casot/ To find potential off targets in any genome FASTA files sgRNA and OT sites User input Free of cost/ First tool that identifies off-targetes in a user specified genome No account for identitiy of mismatches N/A
CRISPR ERA http://crispr-era.stanford.edu/ Used for genome wide screening based on CRISPR, CRISPRi and CRISPRa DNA sequence, gene name or TSS location Candidate guide sequences and distances to TSS 9 Free of cost/can also be used for genome imaging and CRISPR synthetic circuit design   [44]
CCTop http://crispr.cos.uni-heidelberg.de/ It helps in identifying and ranking all sgRNA targets sites according to off target quality DNA sequence/FASTA file single/batch/ sequences 23 to 500 bp Scores OTs and also ranks sgRNA by OTs 45 Free of cost/easy to use No on–target efficacy prediction [45]
WU-CRISPR http://crispr.wustl.edu/ Potential candidate gRNA DNA sequence/gene symbol sgRNA list ranked by efficacy score 2 (Human and mouse) Free of cost/easy to use No account of identity of mismatches [46]
GTscan http://gt-scan.csiro.au/ Used to find target sequences and OTs for a single sequence DNA sequence/FASTA file SgRNA, genomic sites with 0 to 3 mismatches 51 Free of cost/easy to use Trouble in finding exact matches in genome [47]
CRISPR direct https://crispr.dbcls.jp/ To find target sequences for a single transcript/sequence DNA sequence/genome location/transcript Target sequence and position 20 Free of cost/rapid visual display of target sequence and OT information No on-target efficacy [48]
COD http://cas9.wicp.net/ Used to find target sequences for an input sequence DNA sequence up to 400 bp Gene bank file/CSC file/ OT scoring 27 Free of cost/easy to use/ OT scoring Slow to use/no on-taget prediction N/A
sgRNA scorer 2.0 https://crispr.med.harvard.edu/ Used to find target sequences/OT using Casfinder DNA sequence/FASTA files upto10 kb Target sequence with activity score 14 Free of cost/ allows to identify target sites for any CRISPR system Slow to use/OT prediction does not account identity N/A
CRISPOR http://crispor.tefor.net/ To find candidate guide sequences DNA sequence 1000 bp Guide sequence with specificity score/guides for OTs 146 Free of cost N/A [49]