NEW YORK (GenomeWeb) – An international team led by researchers in the US and Japan has developed a new CRISPR method which it claims solves the problem of inefficient targeted DNA cassette insertion in the engineering of animal models.
The team described the method — called Efficient additions with ssDNA inserts-CRISPR or Easi-CRISPR — in Genome Biology today.
Conditional knockout mice and transgenic mice are needed in a large majority of genetic research studies, but creating them involves "labor-intensive methods of homologous recombination in embryonic stem cells and are available for only [about] 25 percent of all mouse genes," the team wrote. "Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient."
To solve this problem, the researchers created a targeting strategy in which long single-stranded DNA donors are combined with pre-assembled complexes that include crRNA, tracrRNA, and Cas9 ribonucleoprotein and are injected into mouse zygotes. "We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5 percent to 100 percent of the resulting live offspring," the authors added.
To test the method, the researchers selected the gene Pitx1 and generated a 1,046-base ssDNA donor containing a floxed version of exon 2, flanked by 93- and 91-base left and right homology arms, respectively. They designed two guide RNAs (sgRNAs) to cut the genome immediately adjacent to each homology arm, and then injected the ssDNA donor with Cas9 mRNA and the two sgRNAs into mouse zygotes.
"Genotyping of the resulting live offspring, using three sets of PCR reactions specific for targeted insertion of each LoxP site and for the entire floxed exon, revealed that one out of eight (13 percent) carried a correctly floxed allele," the researchers said. "Three other pups had partial insertions of the donor cassette: two contained only a single targeted LoxP site and one contained both LoxP insertions, but they were located on separate alleles."
They then prepared "a crRNA + tracrRNA + Cas9 protein complex using chemically synthesized crRNAs and tracrRNAs" designed to cleave Pitx1 in exactly the same sites as the sgRNAs. Two animals out of 10 treated with this method had no insertions, four had partial insertions of a single LoxP site, and four had floxed alleles. The team selected six more genes to generate floxed alleles using Easi-CRISPR and found that the targeting strategy succeeded for all six genes, with efficiencies ranging from 8.5 percent to 100 percent.
"In summary, for all seven genes combined, genotyping of 46 G0 pups showed that 20 (43 percent) contained at least one correctly floxed allele," the researchers added. "Of the 20 founders with correctly floxed exons, two contained point mutations in the inserted regions that may have derived from enzymatic misincorporation during preparation of the ssDNA donor templates. Nevertheless, such mutations did not affect the overall goal of generating floxed mice because we obtained at least one founder with a correct insertion for each gene. Moreover, even the founders with mutations are potentially useful because the mutations were located in intronic sites that are less likely to affect gene function."
Further, when the Easi-CRISPR founder mice were bred to wild-type mice, they transmitted their mutant alleles to their offspring. "These results indicate that Easi-CRISPR can efficiently insert sequences that encode and express reporters, recombinases, and regulatory proteins, and that the technique is applicable to multiple genomic loci," the authors said.
The team also noted its finding that two cleavages can be used to take out a target exon and replace it with a floxed exon cassette, leading to the conclusion that Easi-CRISPR could be used to create gene-replacement models such as a set of point mutations spread across a region, testing regulatory sequences, and replacing short stretches of gene segments or coding sequences from other species. Easi-CRISPR could also be used to modify existing knock-in alleles, the authors added.