Journal
GENOME BIOLOGY
Volume 18, Issue -, Pages -Publisher
BMC
DOI: 10.1186/s13059-017-1220-4
Keywords
CRISPR/Cas9; Homology directed repair; Easi-CRISPR; long ssDNA donors; CRISPR ribonucleoproteins; Cre-LoxP; Conditional knockout; Reporter and recombinase knock-in
Funding
- Institutional Development Award [P20GM103471]
- NIGMS [1P30GM110768-01, P30CA036727]
- NIH [R21DC014779, EY10542]
- KAKENHI from JSPS [26830131, 16 K07085]
- MEXT grants from Nakatani Foundation
- SENSHIN Medical Research Foundation
- Mochida Memorial Foundation
- Takeda Science Foundation
- MRI
- CNSI/NINS [BS281001]
- JSPS [KAKENHI 15 K19988]
- Nakatomi Foundation
- Ichiro
- Kanehara Foundation
- Research and Study Project of Tokai University General Research Organization
- Tokai University School of Medicine
- Ministry of Education, Culture, Sports, Science and Technology (MEXT) [16 K18821]
- MEXT
- Wellcome Trust [087377]
- Fred and Pamela Buffet Cancer Center's ACS Institutional Research Grant
- Research to Prevent Blindness
- Grants-in-Aid for Scientific Research [16H04685, 16K18821, 16K07085, 26830131] Funding Source: KAKEN
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Background: Conditional 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 similar to 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. Results: Here 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.
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