Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice

Tomomi Aida, Keiho Chiyo, Takako Usami, Harumi Ishikubo, Risa Imahashi, Yusaku Wada, Kenji Tanaka, Tetsushi Sakuma, Takashi Yamamoto, Kohichi Tanaka

Research output: Contribution to journalArticle

128 Citations (Scopus)

Abstract

Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools.

Original languageEnglish
Article number87
JournalGenome Biology
Volume16
Issue number1
DOIs
Publication statusPublished - 2015 Apr 29

Fingerprint

CRISPR-Cas Systems
RNA
Organism Cloning
molecular cloning
Guide RNA
mice
Knockout Mice
digestion
Digestion
genomics
Messenger RNA
protein
Research
Proteins
cloning
proteins

ASJC Scopus subject areas

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

Cite this

Aida, T., Chiyo, K., Usami, T., Ishikubo, H., Imahashi, R., Wada, Y., ... Tanaka, K. (2015). Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biology, 16(1), [87]. https://doi.org/10.1186/s13059-015-0653-x

Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. / Aida, Tomomi; Chiyo, Keiho; Usami, Takako; Ishikubo, Harumi; Imahashi, Risa; Wada, Yusaku; Tanaka, Kenji; Sakuma, Tetsushi; Yamamoto, Takashi; Tanaka, Kohichi.

In: Genome Biology, Vol. 16, No. 1, 87, 29.04.2015.

Research output: Contribution to journalArticle

Aida, T, Chiyo, K, Usami, T, Ishikubo, H, Imahashi, R, Wada, Y, Tanaka, K, Sakuma, T, Yamamoto, T & Tanaka, K 2015, 'Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice', Genome Biology, vol. 16, no. 1, 87. https://doi.org/10.1186/s13059-015-0653-x
Aida T, Chiyo K, Usami T, Ishikubo H, Imahashi R, Wada Y et al. Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biology. 2015 Apr 29;16(1). 87. https://doi.org/10.1186/s13059-015-0653-x
Aida, Tomomi ; Chiyo, Keiho ; Usami, Takako ; Ishikubo, Harumi ; Imahashi, Risa ; Wada, Yusaku ; Tanaka, Kenji ; Sakuma, Tetsushi ; Yamamoto, Takashi ; Tanaka, Kohichi. / Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. In: Genome Biology. 2015 ; Vol. 16, No. 1.
@article{057f2cfb423f44e9ac004799b233e6a3,
title = "Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice",
abstract = "Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50{\%} efficiency, compared with just 10{\%} by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools.",
author = "Tomomi Aida and Keiho Chiyo and Takako Usami and Harumi Ishikubo and Risa Imahashi and Yusaku Wada and Kenji Tanaka and Tetsushi Sakuma and Takashi Yamamoto and Kohichi Tanaka",
year = "2015",
month = "4",
day = "29",
doi = "10.1186/s13059-015-0653-x",
language = "English",
volume = "16",
journal = "Genome Biology",
issn = "1474-7596",
publisher = "BioMed Central",
number = "1",

}

TY - JOUR

T1 - Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice

AU - Aida, Tomomi

AU - Chiyo, Keiho

AU - Usami, Takako

AU - Ishikubo, Harumi

AU - Imahashi, Risa

AU - Wada, Yusaku

AU - Tanaka, Kenji

AU - Sakuma, Tetsushi

AU - Yamamoto, Takashi

AU - Tanaka, Kohichi

PY - 2015/4/29

Y1 - 2015/4/29

N2 - Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools.

AB - Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools.

UR - http://www.scopus.com/inward/record.url?scp=84938945636&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84938945636&partnerID=8YFLogxK

U2 - 10.1186/s13059-015-0653-x

DO - 10.1186/s13059-015-0653-x

M3 - Article

C2 - 25924609

AN - SCOPUS:84938945636

VL - 16

JO - Genome Biology

JF - Genome Biology

SN - 1474-7596

IS - 1

M1 - 87

ER -