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Review
, 199 (1), 1-15

A Mouse Geneticist's Practical Guide to CRISPR Applications

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Review

A Mouse Geneticist's Practical Guide to CRISPR Applications

Priti Singh et al. Genetics.

Abstract

CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Even for the laboratory mouse, a model that has thrived under the benefits of embryonic stem (ES) cell knockout capabilities for nearly three decades, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 technology enables one to manipulate the genome with unprecedented simplicity and speed. It allows generation of null, conditional, precisely mutated, reporter, or tagged alleles in mice. Moreover, it holds promise for other applications beyond genome editing. The crux of this system is the efficient and targeted introduction of DNA breaks that are repaired by any of several pathways in a predictable but not entirely controllable manner. Thus, further optimizations and improvements are being developed. Here, we summarize current applications and provide a practical guide to use the CRISPR/Cas9 system for mouse mutagenesis, based on published reports and our own experiences. We discuss critical points and suggest technical improvements to increase efficiency of RNA-guided genome editing in mouse embryos and address practical problems such as mosaicism in founders, which complicates genotyping and phenotyping. We describe a next-generation sequencing strategy for simultaneous characterization of on- and off-target editing in mice derived from multiple CRISPR experiments. Additionally, we report evidence that elevated frequency of precise, homology-directed editing can be achieved by transient inhibition of the Ligase IV-dependent nonhomologous end-joining pathway in one-celled mouse embryos.

Keywords: CRISPR; genome editing; mouse knockouts; nonhomologous end joining.

Figures

Figure 1
Figure 1
Schematic showing the proposed cellular repair pathways operating at CRISPR/Cas9-generated DNA breaks (A) or nicks (B). (A) gRNA targeted Cas9 having HNH and RuvC domains induces a DNA break on complementary and noncomplementary strands, respectively. These DSBs may be repaired predominantly by the less error-prone C-NHEJ pathway (I). If C-NHEJ fails, unrepaired DSB sites are recognized by PARP1 thus entering the alt-NHEJ (II) pathway. The Ku-unprotected DNA ends are resected and ultimately ligated by either Ligase III or Ligase I, thus generating longer indels at targeted loci. Alternatively, presence of donor template (ssODN or dsODN) carrying designed mutation (yellow box) may promote homology-directed repair (III) leading to precise editing. Although the exact mechanism of DNA repair using ssODNs is still unknown, CRISPR/Cas9-mediated precise editing with ssODNs is relatively efficient. (B) Cas9 nickase (Cas9D10A), bearing a mutation in RuvC nuclease domain, cleaves the DNA strand complementary to gRNA. The nick is predominantly repaired by the error-free BER pathway or simply undergoes nick ligation (I). In the presence of a ssODN, the nick may also be repaired by BRCA1-dependent HDR (II), generating a precise mutation.
Figure 2
Figure 2
Sequencing-based methods to identify CRISPR-edited alleles in founder mice. (A) Sanger sequencing of PCR products around gRNA binding site. PCR amplification from mouse tail biopsy DNA will generate a mixture of two or more (mosaic) amplicons representing allelic variants in the mouse. This can cause overlapping peaks on the chromatogram (red arrow) and difficulty in identifying the mutation(s). (B) Sequencing of plasmid-cloned PCR products. Each clone contains one amplicon/allelic variant present in a mouse. This requires sequencing at least 10 single colonies per targeting event per mouse (e.g., one gene × 20 founder mice × 10 colonies = 200 sequences). In the case of multiplexed editing, proportionately more clones must be sequenced. (C) Next-Gen-based multiplexed sequencing. This method also allows testing for off-target (OT) events and the presence of mosaicism. Target and OT PCR products from one founder mouse are labeled with unique barcode. All PCR products from up to 96 mice (one mouse = one barcode) are pooled together and sequenced. *, mosaic animal.
Figure 3
Figure 3
Transient inhibition of C-NHEJ with the Ligase IV inhibitor (SCR7) increases editing efficiency. In the presence of SCR7, DSBs will be predominantly repaired by the highly error-prone alt-NHEJ (II) pathway, generating indels, or HDR-mediated precise editing (yellow box) (III). Thickness of the arrows represents relative interplay of individual pathways involved in the repair of targeted DSB in presence of SCR7.

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