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  • A Mouse Geneticist’s Practical Guide to CRISPR Applications
    Priti Singh, John C. Schimenti, Ewelina Bolcun-Filas
    Genetics January 2015 199: 1-15; https://doi.org/10.1534/genetics.114.169771
    ...uses a homologous template, usually a sister chromatid under natural circumstances, to repair DNA damage if DNA replication has already occurred. Therefore, codelivering the site-specic nuclease with an alternative repair template, such as a plasmid or single-stranded oligodeoxynucleotide (ss ~~~
  • Open Access
    A Mouse Geneticist’s Practical Guide to CRISPR Applications
    Priti Singh, John C. Schimenti, Ewelina Bolcun-Filas
    Genetics Jan 2015, 199 (1) 1-15; DOI: 10.1534/genetics.114.169771
    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.
  • Open Access
    A Mouse Geneticist’s Practical Guide to CRISPR Applications
    Priti Singh, John C. Schimenti, Ewelina Bolcun-Filas
    Genetics Jan 2015, 199 (1) 1-15; DOI: 10.1534/genetics.114.169771
    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.
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