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

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]

[143]

CRISPR-ERA

Analyze gene editing and gene regulation

Sequence starts with

N20NGG

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.

3

CRISPR/Cas9

NGG

NAG

[144]

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

NAG

[145]

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

NAG

[147]

CRISPy-web

Design sgRNAs

API

antiSMASH

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

NAG

[149]

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]

[153]

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]