Detailed Phenotypic and Molecular Analyses of Genetically Modified Mice Generated by CRISPR-Cas9-Mediated Editing. Bijal A. Parikh, Diana L.Beckman, Swapneel J. Patel, J. Michael White, Wayne M. Yokoyama. PLOS ONE |DOI:10.1371/journal.pone.0116484 January14, 2015
It's always tough to generate lots of numbers across mouse injections. It's just a lot of work and expensive. Despite some small numbers in the individual experiments, and some repeat description of indel mutations, etc. that others have reported, this group presents some new insights that are good to know. The most important one I got from this paper was an interesting observation concerning their gene repair experiments.
The target gene in this case was Tyr, or Tyrosinase, which of course when completely inactivated causes albinism. This group did several good comparisons of different targets, nickase vs. "native" Cas9, and also injections on C57BL6 ("B6")versus albino C57BL6 ("AB6"). Thus, they could make induce mutations disrupting wild type Tyr in the B6 strain - generating albino mice - while attempting to repair the already-mutated Tyr in the albino AB6 strain.
To do the latter they tried using as the donor a wild-type ds-DNA fragment with 800 bp homology arms flanking the wild type region of the CRISPR target(s). This failed to repair Tyr in AB6 mice, which in of itself was not a tragedy - CRISPR-mediated repair is not as efficient as simple mutagenesis and the sample size was low. But what they also observed is that these injections generated zero indel mutations, even though they showed the same CRISPR reagents generated lots of mutations at the target sites just fine when they were injected WITHOUT the wild type donor DNA molecule. Therefore they repeated the experiments with a modified donor DNA fragment that had sense mutations disrupting the CRISPR target site. This worked well in both generating a few correctly repaired alleles, but also restoring high rates of indels generated by NHEJ, thus proving that the wild-type molecule itself was inhibiting the CRISPR machinery. Since the donor was a double-stranded DNA, it makes sense that it was "soaking up" the Cas9/sgRNA, It was certainly present at large stoichiometric excess to the chromosomal targets.
I counted up the gene repair injections they did with either the WT or modified donor DNA fragment, and the summary was as follows:
Experiment : (Mice with indels/ Mice with HDR-mediated repair / Total number mice)
Wild-type donor DNA: 0 / 0 / 27. Across 3 experiments,
Modified donor DNA: 18 / 3 / 41. Across 5 experiments.
These experiments were a mix of nickase vs. native Cas9, 2 different targets, and B6 and AB6 mice. But the trend seems like it's clear - CRISPR-mediated mutations were strongly inhibited with a wild-type donor DNA that carried a perfect match to the CRISPR target.
Would a single-stranded donor oligo with a wild-type target match also inhibit? It's not clear to me, as I think all the oligo edits I've seen published disrupted the target so that doesn't provide data; the single stranded DNA probably won't complex with Cas9.