@article{1b424def45c94810a4e1ae3244434181,
title = "Easi-CRISPR: A robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins",
abstract = "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 ~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.",
keywords = "CRISPR ribonucleoproteins, CRISPR/Cas9, Conditional knockout, Cre-LoxP, Easi-CRISPR, Homology directed repair, Long ssDNA donors, Reporter and recombinase knock-in",
author = "Quadros, {Rolen M.} and Hiromi Miura and Harms, {Donald W.} and Hisako Akatsuka and Takehito Sato and Tomomi Aida and Ronald Redder and Richardson, {Guy P.} and Yutaka Inagaki and Daisuke Sakai and Buckley, {Shannon M.} and Parthasarathy Seshacharyulu and Batra, {Surinder K.} and Behlke, {Mark A.} and Zeiner, {Sarah A.} and Jacobi, {Ashley M.} and Yayoi Izu and Thoreson, {Wallace B.} and Urness, {Lisa D.} and Mansour, {Suzanne L.} and Masato Ohtsuka and Gurumurthy, {Channabasavaiah B.}",
note = "Funding Information: We thank Harumi Ishikubo, Takako Usami, and the genome editing facility at laboratory of recombinant animals, MRI, TMDU, and Y. Wada (FASMAC) for providing technical assistance and materials. We thank Adrian Koesters of UNMC for her editorial contributions and Joseph M. Miano, University of Rochester for his helpful comments on the manuscript. We also gratefully acknowledge the contribution of the staff of the Support Center for Medical Research and Education, Tokai University, for sequencing and microinjection. Funding This work was supported in part by an Institutional Development Award (PI: Shelley Smith) P20GM103471 (to CBG, RMQ, DWH, and RR), by NIGMS 1P30GM110768-01 and P30CA036727 to RR (as part of support to University of Nebraska DNA Sequencing Core), by NIH R21DC014779 (to SLM) and by KAKENHI (26830131, 16 K07085, Comprehensive Brain Science Network and Adaptive Circuit Shift) from JSPS and MEXT, grants from Nakatani Foundation, SENSHIN Medical Research Foundation, Mochida Memorial Foundation, Takeda Science Foundation, MRI, and CNSI/NINS (BS281001) to TA, KAKENHI 15 K19988 from JSPS and grants from the Nakatomi Foundation, the Ichiro, Kanehara Foundation, and Takeda Science Foundation to Y. Izu, and by Research and Study Project of Tokai University General Research Organization, 2016-2017 Tokai University School of Medicine Project Research to MO, and by Grant-in- Aid for Young Scientists (B) (16 K18821) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to HM, and by MEXT-Supported Program for the Strategic Research Foundation at Private Universities 2015-2019 to Tokai University, and the Wellcome Trust (grant 087377) to GPR, Fred and Pamela Buffet Cancer Center's ACS Institutional Research Grant to SMB, by NIH grant EY10542 and Senior Scientific Investigator Award Research from Research to Prevent Blindness (to WBT).",
year = "2017",
month = may,
day = "17",
doi = "10.1186/s13059-017-1220-4",
language = "English (US)",
volume = "18",
journal = "Genome Biology",
issn = "1465-6906",
publisher = "BioMed Central",
number = "1",
}