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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 [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   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   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   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
  1. 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
  2. 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