Table 1.
Tools for the design of guide RNAs
| Tool | Website | Reference | Input a | Output b | Throughput c | Use cases | Genomes | sgRNA sequence constraints | Validation | Pros | Cons | Software |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| sgRNA Designer | http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design | [37] | Ensembl transcript IDs or nucleotide sequences | Activity-ranked sgRNA, exon, percentage protein sequence C-terminal of target site | Medium/high | Find target sequences for up to ten transcripts (small-batch mode); good for generating a lot of candidate guides quickly | Human, mouse | N/A | Meta-analysis of genome-wide CRISPR screens | Ease of use, on-target efficacy based on experiment data and validation | No OT prediction | Web/local |
| CRISPR MultiTargeter | http://www.multicrispr.net/ | [50] | Gene/transcript ID or sequence | Activity-ranked sgRNA based on sgRNA Designer (see above). Reports percentage GC and Tm | Low | Find all target sequences for a single sequence; find all common target sequences for all transcripts for a given gene; find unique/non-unique target sequences providing multiple sequences or similar gene types | 12 common genomes | 5′ G or GG. Target length. PAM NGG or user specified. Paired sgRNA | Ease of use. Many options, including any PAM. Has multiple modes, separated out, that could be useful | No OT prediction. Multiple modes can be complicated | Web | |
| Cas9 Design | http://cas9.cbi.pku.edu.cn | [102] | Input sequence or FASTA file | Target sequence and exact matches in reference genome. Percentage AT, predicted RNA folding structure for sgRNA | Low | Find target sequences for a single sequence | Ten common genomes | Target length. User-specified scaffold RNA for structural predictions | Can be used to identify potentially problematic hairpin structures in sgRNAs | No on-target efficacy prediction. No OT prediction | Web | |
| SSFinder | https://code.google.com/p/ssfinder/ | [46] | FASTA file | All potential NGG-PAM guides | High | Find target sequences for any number of FASTA sequences | N/A | None | Very simple input/output requirements. Works quickly on a small number of sequences. High throughput possible | No options. No on-target or off-target information | Python script | |
| Cas OFFinder | http://www.rgenome.net/cas-offinder/ | [103] | sgRNA | Lists OT sites with number and position of MM | Medium/high | Find comprehensive off-target information for one or more guide sequences; must run as script | Approximately 20 common genomes | Alternative PAMs, tolerance for MMs | Ease of use. Analyzes multiple sgRNAs in batch. OT sites with up to nine MMs and two bulges | Does not indicate whether OTs are in CDS. Does not account for identity of MMs | Web/local |
aThe input data are gene sequence or sgRNA sequence. bThe output is sgRNA sequence or off-target sites. c‘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
Abbreviations: CDS coding sequence,CRISPR clustered regularly interspaced short palindromic repeats, MM mismatch, N/A not applicable, OT off-target, PAM protospacer adjacent motif, sgRNA single guide RNA, Tm melting temperature