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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 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 Candidate gRNA DNA sequence/gene name Candidate guidesequences and off target loci 5 Free of cost   N/A
CRISPR design 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 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 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 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 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 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 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 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 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 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 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 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 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 To find candidate guide sequences DNA sequence 1000 bp Guide sequence with specificity score/guides for OTs 146 Free of cost N/A [49]