Table 1 Tools for the design of guide RNAs
From: Resources for the design of CRISPR gene editing experiments
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 | [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 | [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 | Â | Can be used to identify potentially problematic hairpin structures in sgRNAs | No on-target efficacy prediction. No OT prediction | Web | |
SSFinder | [46] | FASTA file | All potential NGG-PAM guides | High | Find target sequences for any number of FASTA sequences | N/A | None | Â | 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 | [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 | Â | 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 |