CRISPR Guide RNA Validation In Vitro

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been applied to edit genomes in a wide variety of model systems. Although this process can be quite efficient, editing at precise locations in the genome remains difficult without a suitable single guide RNA (sgRNA). We have developed a method for screening sgRNA function in vitro, using reagents that most zebrafish laboratories are already using. The results from our in vitro assay correlate with function in vivo in every sgRNA that we have examined so far. When combined with endonucleases with alternative protospacer adjacent motif site specificities and alternative sgRNAs, this method will streamline genome editing at almost any locus.

Keywords: CRISPR; Cas9; in vitro; knock in; validation; zebrafish.

FIG. 1.

Guide RNA screening of the tyrosinase locus. (A) Schematic of tyrosinase gene structure. The tyrosinase gene is encoded by five exons. The sequence encoding the enzymatic Cu-binding domain is highlighted in purple. The CHOPCHOP algorithm was used to predict the top 18 sgRNA binding sites that began with GG, the positions of which are indicated with arrowheads. The CHOPCHOP confidence scores are indicated as high (green), medium (yellow), or low (red). The positions of sgRNAs validated in vivo are represented with larger triangles and their CHOPCHOP rank number. (B) Schematic of assay to detect CRISPR/Cas9-mediated cleavage in vitro. Sequences surrounding the target site were PCR amplified from genomic DNA and combined with Cas9 proteins and sgRNA; reactions were allowed to proceed and run out on a gel to detect cleavage. (C) Summary of CHOPCHOP rank (1–18; 1 is best), CRISPRscan score (1–100; 100 is best), in vitro assay results, in vivo T7EI assay results, exon, strand, and GC content (confidence scores are indicated as high (green), medium (yellow), or low (red)). Note that there was no significant correlation with reactivity in vitro and CHOPCHOP rank, CRISPRscan score, strand, or GC content. n/a sgRNAs were not tested in vivo. (D) Sample from chosen sgRNAs with Cas9 assay run next to in vivo T7E1 assay. (E). Zebrafish at 48 h postfertilization after injection with chosen guides. Note the loss of melanocytes compared with the uninjected control in rank 2 and 11. There was no change in melanocyte distribution and coloration in rank 6 and rank 9 injected fish. CRISPR, clustered regularly interspaced short palindromic repeats; PCR, polymerase chain reaction; sgRNA, single guide RNA.