Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins

Abstract

BackgroundConditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. 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.ResultsHere we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5–100% of the resulting live offspring.Conclusions Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1220-4) contains supplementary material, which is available to authorized users.

CRISPR/Cas9-Facilitated Chromosome Engineering to Model Human Chromosomal Alterations

“…Therefore, zygote injection of Cas9 protein instead of the mRNA may help to reduce mosaicism in founders [21], because Cas9 protein-RNA complex is more likely to degrade rapidly, leading to a shorter half-life than Cas9 mRNA [24][25][26][27]. The results from some recent studies support such reasoning [18][19][20]26].…”

“…Unlike sgRNA, in dual-crRNA:tracrRNA, crRNA and tracrRNA are synthesized separately, and then annealed together to be used as the guide RNA [15,18,19]. Recently, a few reports have demonstrated that a dual-crRNA:tracrRNA combined with Cas9 protein can increase the eiciency of genome editing, especially the frequency of HDR [18][19][20]. Therefore, dual-crRNA:tracrRNA should still be considered when planning chromosome rearrangement projects.…”

Beyond editing to writing large genomes

“…SSOs alone have demonstrated efficiencies ranging from 10 −5 to 2% in mammalian cells (Table 1) 33–35 . However, the most prevalent utility of SSOs are in conjunction with a designer nuclease to generate much higher rates of modification 5,36 .…”

The fourth annual BRDS on genome editing and silencing for precision medicines

“…Similarly, Dr. Rolf Turk from Integrated DNA Technologies (IDT) talked about the insertion of targeted repair template for the generation of knock-in strains or animal models, including insertion of reporter genes or specific mutations. As an evident, when the repair template consists of double-stranded DNA, the knock-in efficiency is usually very low [26]. Therefore, to overcome this low knock-in frequency, the use of single-stranded oligonucleotides could be an alternative as HDR templates.…”

“…However, the generation of specific disease models in animals that recapitulate the biological and clinical characteristics of humans by targeting specific locus in the genome has made CRISPR-Cas system an essential subject for studying various diseases [29]. Dr. Channabasavaiah Gurumurthy from UNMC, introduced Easi- CRISPR ( E fficient a dditions with s sDNA i nserts-CRISPR), a strategy of using long single-stranded DNA as a donor template to solve the major issues related to animal genome engineering by the inefficiency of targeted DNA cassette insertion [26]. Per.…”