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

Rolen M. Quadros, Hiromi Miura, Donald W. Harms, Hisako Akatsuka, Takehito Sato, Tomomi Aida, Ronald Redder, Guy P. Richardson, Yutaka Inagaki, Daisuke Sakai, Shannon M Buckley-McKeown, Parthasarathy Seshacharyulu, Surinder Kumar Batra, Mark A. Behlke, Sarah A. Zeiner, Ashley M. Jacobi, Yayoi Izu, Wallace B Thoreson, Lisa D. Urness, Suzanne L. MansourMasato Ohtsuka, Channabasavaiah B Gurumurthy

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Article number92
JournalGenome biology
Volume18
Issue number1
DOIs
StatePublished - May 17 2017

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
ribonucleoproteins
single-stranded DNA
Ribonucleoproteins
allele
Alleles
Zygote
alleles
mice
zygote
targeting
Knockout Mice
Transgenic Mice
DNA
methodology
Recombinases
genetically modified organisms
recombination
Single-Stranded DNA
Homologous Recombination

Keywords

  • CRISPR ribonucleoproteins
  • CRISPR/Cas9
  • Conditional knockout
  • Cre-LoxP
  • Easi-CRISPR
  • Homology directed repair
  • Long ssDNA donors
  • Reporter and recombinase knock-in

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Genetics
  • Cell Biology

Cite this

Easi-CRISPR : A robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. / Quadros, Rolen M.; Miura, Hiromi; Harms, Donald W.; Akatsuka, Hisako; Sato, Takehito; Aida, Tomomi; Redder, Ronald; Richardson, Guy P.; Inagaki, Yutaka; Sakai, Daisuke; Buckley-McKeown, Shannon M; Seshacharyulu, Parthasarathy; Batra, Surinder Kumar; Behlke, Mark A.; Zeiner, Sarah A.; Jacobi, Ashley M.; Izu, Yayoi; Thoreson, Wallace B; Urness, Lisa D.; Mansour, Suzanne L.; Ohtsuka, Masato; Gurumurthy, Channabasavaiah B.

In: Genome biology, Vol. 18, No. 1, 92, 17.05.2017.

Research output: Contribution to journalArticle

Quadros, RM, Miura, H, Harms, DW, Akatsuka, H, Sato, T, Aida, T, Redder, R, Richardson, GP, Inagaki, Y, Sakai, D, Buckley-McKeown, SM, Seshacharyulu, P, Batra, SK, Behlke, MA, Zeiner, SA, Jacobi, AM, Izu, Y, Thoreson, WB, Urness, LD, Mansour, SL, Ohtsuka, M & Gurumurthy, CB 2017, 'Easi-CRISPR: A robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins', Genome biology, vol. 18, no. 1, 92. https://doi.org/10.1186/s13059-017-1220-4
Quadros, Rolen M. ; Miura, Hiromi ; Harms, Donald W. ; Akatsuka, Hisako ; Sato, Takehito ; Aida, Tomomi ; Redder, Ronald ; Richardson, Guy P. ; Inagaki, Yutaka ; Sakai, Daisuke ; Buckley-McKeown, Shannon M ; Seshacharyulu, Parthasarathy ; Batra, Surinder Kumar ; Behlke, Mark A. ; Zeiner, Sarah A. ; Jacobi, Ashley M. ; Izu, Yayoi ; Thoreson, Wallace B ; Urness, Lisa D. ; Mansour, Suzanne L. ; Ohtsuka, Masato ; Gurumurthy, Channabasavaiah B. / Easi-CRISPR : A robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. In: Genome biology. 2017 ; Vol. 18, No. 1.
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AU - Miura, Hiromi

AU - Harms, Donald W.

AU - Akatsuka, Hisako

AU - Sato, Takehito

AU - Aida, Tomomi

AU - Redder, Ronald

AU - Richardson, Guy P.

AU - Inagaki, Yutaka

AU - Sakai, Daisuke

AU - Buckley-McKeown, Shannon M

AU - Seshacharyulu, Parthasarathy

AU - Batra, Surinder Kumar

AU - Behlke, Mark A.

AU - Zeiner, Sarah A.

AU - Jacobi, Ashley M.

AU - Izu, Yayoi

AU - Thoreson, Wallace B

AU - Urness, Lisa D.

AU - Mansour, Suzanne L.

AU - Ohtsuka, Masato

AU - Gurumurthy, Channabasavaiah B

PY - 2017/5/17

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N2 - 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.

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KW - Cre-LoxP

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KW - Homology directed repair

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KW - Reporter and recombinase knock-in

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