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Table 3 Online bioinformatics tools detect the accurate editing site within the whole genome and evade off-targeting percentage probability

From: Strategies to overcome the main challenges of the use of CRISPR/Cas9 as a replacement for cancer therapy

Tool name Description Input Output Maximum mismatches allowed Supported nucleases PAM sequence References
CRISPResso2 Genome editing
and interpretation of amplicon sequencing
1. Editing of tool specification.
2. Input sequences
3. Amplicon sequence, sgRNA sequence
1. indel sizes and positions
2. HDR/NHEJ frequency
3. sequence alignment with reference
4. allele-specific quantification
- Cas9 Cpf1 NGS (139)
Cas-Analyzer Genome editing and programmable nucleases 1. Fastq
2. gzip-compressed
1. indel sizes and positions
2. HDR/NHEJ frequency
3. sequence alignment with reference
up to a 1-nt SpCas9, StCas9, NmCas9, SaCas9, CjCas9, AsCpf1/LbCpf1, paired nucleases: ZFNs, TALENs, Cas9 nickases, dCas9-FokI NGS [140]
CRISPR-GA Quantification of the edited site then analysis of the different alterations. Paired-end reads 1. indel sizes and positions
2. HDR/NHEJ frequency
< 20 Cas9 NGS [141]
TIDE/TIDER Identification of major induced mutations in the editing site using specially developed decomposition algorithm DNA from a pool of cells treated with RGEN Cas9) and a character string representing the sgRNA sequence (20 nt) 1. indel sizes and positions
2. HDR/NHEJ frequency
1% SpCas9, SaCas9, St1Cas9, NmCas9, AsCpf1, FnCpf1, LbCpf1 Sanger sequencing [142]
CRISPR-ERA Analyze gene editing and gene regulation Sequence starts with
1. gRNA design
2. E score (efficacy score)
3. S score ( specificity score)
4. E+S score: the sum of efficacy score and Specificity score.
CRISPRseek Using various tools for the CRISPR editing
including Base Editors and the Prime Editor for input target sequences,
The RNAs sequence is annotated with a total score of the top5 and topN off-targets and Cas9 1. gRNA design
2. off-targeting count
3. score on targeting
4. find Spacer
4 User customizable NGG
CHOPCHOP v3 Web tool for selecting alternative transcription of RNA using CRISPER-CAS13 gene name, genomic coordinates, or a pasted sequence (including RefSeq and ENSEMBL gene IDs) 1. gRNA design, Off-targeting
2. GC content (%)
3. number of self-complementary
4. efficiency
- CRISPR effector (e.g., Cas9, CasX, or Cas13) NGG [146]
E-CRISP Specific Algorithm used to target any nucleotide sequence ranging from single exons to entire genomes FASTA 1. gRNA design (for various targeting purposes)
2. gene annotation filtering
3. off-targeting analysis.
4. image for (genomic context, restriction site, TSS, strop, and start Condon)
- genomic context (e.g., exons, transcripts, CpG islands) NGG
CRISPy-web Design sgRNAs API
gRNA design, Target Site selection 3 Cas9 NGG [148]
CRISPR-P 2.0 Genome editing in plant Gene name, ID, position, and sequence 1. on-target score
2. off-target score
3. GC content
4. restriction endonuclease site
- Cas9 NGG
COSMID Validation and identification of off-target sequence FetchGWI search program Off-target score
GC content (%)
3 CRISPR Off-target Sites with Mismatches, Insertions, and Deletions NGG, NAG, NRG [150]
WU-CRISPR Gene editing and detection of CRISPR/Cas9 Knockout Gene Sequence in FASTA format 1. gRNA sequence
2. potency score
3. off-target status
4. BLAST alignment
5. coding sequence
-   NGG, [151]
Cas-Designer Selecting all RGEN targets via Microhomology-predictor FASTA 1. RGENs
2. Cas-OFFinder
3. Cas-Designer
4. Cas-Database.
0-10 NGG, NRG, NNAGAAW, NNNNGMTT It depends on the Cas protein. [152]
CRISPR MultiTargeter Web program to detect the High identical site in multiple genes FASTA Multiple sequence Alignment 0-24 1. SpCas9 (PAM ‘NGG’),
2. StCas9 (PAM ‘NNAGAAW’),
3. NmCas9 (PAM ‘NNNNGMTT’)
NGG [154]