CRISPRtools: a flexible computational platform for performing CRISPR/Cas9 experiments in the mouse

Abstract

Genome editing using the CRISPR/Cas9 RNA-guided endonuclease system has rapidly become a driving force for discovery in modern biomedical research. This simple yet elegant system has been widely used to generate both loss-of-function alleles and precision knock-in mutations using single-stranded donor oligonucleotides. Our CRISPRtools platform supports both of these applications in order to facilitate the use of CRISPR/Cas9. While there are several tools that facilitate CRISPR/Cas9 design and screen for potential off-target sites, the process is typically performed sequentially on single genes, limiting scalability for large-scale programs. Here, the design principle underlying gene ablation is based upon using paired guides flanking a critical region/exon of interest to create deletions. Guide pairs are rank ordered based upon published efficiency scores and off-target analyses, and reported in a concise format for downstream implementation. The exon deletion strategy simplifies characterization of founder animals and is the strategy employed for the majority of knockouts in the mouse. In proof-of-principle experiments, the effectiveness of this approach is demonstrated using microinjection and electroporation to introduce CRISPR/Cas9 components into mouse zygotes to delete critical exons.

Keywords: Cas9 Protein; Efficiency Score; Exon Deletion; Genome Editing; Nonsense Mediate Decay.

Figure 1

Hypothetical gene models illustrating deletion strategy for producing null alleles using paired CRISPR guides flanking the region of interest. (a) Two examples of internal exon deletions are shown to indicate removal of large portion of coding sequence for single exon gene. The other example shows an internal deletion within an exon for a multi-exon gene that has all exons in the same phase. (b). Paired guides flank a critical exon within a multi-exon gene that when deleted will result in a frameshift mutation. Exon phases (0,1,2) are indicated above the gene model for multi-exon genes and guide cut sites are shown as arrows.

Figure 2

CRISPRtools workflow for designing null alleles. (a) Critical exon identifiers or genomic coordinates are used as input and rank ordered guides are provided as output with a series of summary files containing guide positions, scores, expected deletion sizes, oligo information, and a bed file for visualization on genome browser. (b) Stylized representation of UCSC genome browser view illustrating exon deletion strategy using four guides.

Figure 3

Strategy for generating exon deletions in mouse zygotes. (a) Individual oligos containing T7 promoter followed by the guide sequence are pooled with a universal reverse primer for parallel amplification via PCR. The DNA template pool is subsequently purified and used as input for in vitro transcription reaction. (b) Schematic showing guide positions relative to critical exon (black box) for selected target genes. Paired guides flanking the critical exon are shown above with PAM colored as red to indicate orientation. The expected maximal deletion is shown below as a dashed line. (c) Comparison of delivery method and Cas9 biological form into C57BL/6NJ zygotes. (d) Representative PCR genotyping results for Neil2 showing increased deletion frequency with increased pulse number. Two B6 control DNA samples and no template control (ntc) are included.