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Genome Biol. 2017 May 17;18(1):92. doi: 10.1186/s13059-017-1220-4.

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

Author information

1
Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA.
2
Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
3
Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
4
Department of Host Defense Mechanism, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
5
Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo, Tokyo, 113-8510, Japan.
6
Laboratory of Recombinant Animals, MRI, TMDU, 2-3-10, 2-3-10, Surugadai, Kanda, Chiyoda, Tokyo, 101-0062, Japan.
7
Present address: McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
8
High-Throughput DNA Sequencing and Genotyping Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA.
9
Sussex Neuroscience, University of Sussex, Falmer, Brighton, BN1 9QG, UK.
10
The Institute of Medical Sciences, Tokai University, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
11
Department of Regenerative Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
12
Department of Orthopaedic Surgery, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
13
Department of Genetics, Cell Biology & Anatomy, College of Medicine, University of Nebraska Medical Center, Omaha, NE, USA.
14
Fred and Pamela Buffett Cancer Center, Eppley Institute for Research in Cancer and Allied Disease, University of Nebraska Medical Center, Omaha, NE, USA.
15
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.
16
Integrated DNA Technologies, Inc., Coralville, IA, 52241, USA.
17
Department of Animal Risk Management, Chiba Institute of Science, 3 Shiomi-cho, Choshi, Chiba, 288-0025, Japan.
18
Truhlsen Eye Institute and Department of Ophthalmology & Visual Sciences, University of Nebraska Medical Center, Omaha, NE, 68198, USA.
19
Department of Human Genetics, University of Utah, Salt Lake City, UT, 84112, USA.
20
Department of Human Genetics, University of Utah, Salt Lake City, UT, 84112, USA. suzi.mansour@genetics.utah.edu.
21
Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan. masato@is.icc.u-tokai.ac.jp.
22
Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan. masato@is.icc.u-tokai.ac.jp.
23
The Institute of Medical Sciences, Tokai University, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan. masato@is.icc.u-tokai.ac.jp.
24
Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA. cgurumurthy@unmc.edu.
25
Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center, Omaha, NE, USA. cgurumurthy@unmc.edu.

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; Reporter and recombinase knock-in; long ssDNA donors

PMID:
28511701
PMCID:
PMC5434640
DOI:
10.1186/s13059-017-1220-4
[Indexed for MEDLINE]
Free PMC Article

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