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CLICK: one-step generation of conditional knockout mice.

Yoshiki Miyasaka1, Yoshihiro Uno1, Kazuto Yoshimi2,3

  • 1Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan.

BMC Genomics
|May 4, 2018
PubMed
Summary
This summary is machine-generated.

We developed CLICK, a CRISPR method using long single-stranded DNA, to efficiently create floxed alleles in mice and rats. This technique enables rapid generation of conditional knockout models for genetic research.

Keywords:
CLICKCRISPR/Cas9Cre-loxP systemLong single-stranded DNAZygote electroporation

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Area of Science:

  • Molecular Biology
  • Genetics
  • Gene Editing

Background:

  • CRISPR/Cas9 technology allows precise gene targeting in zygotes.
  • Efficient generation of loxP-flanked (floxed) alleles remains a challenge in genetic engineering.

Purpose of the Study:

  • To develop a rapid and efficient method for generating floxed alleles in zygotes.
  • To establish a streamlined approach for creating conditional knockout models.

Main Methods:

  • Electroporation of Cas9, two guide RNAs (gRNAs), and long single-stranded DNA (lssDNA) into zygotes.
  • The method is termed CRISPR with lssDNA inducing conditional knockout alleles (CLICK).

Main Results:

  • CLICK enables the rapid generation of floxed alleles in both mice and rats.
  • High efficiency of homozygous knock-ins was achieved in oocytes.

Conclusions:

  • CLICK facilitates the one-step generation of conditional knockouts in founder (F0) animals.
  • This method streamlines the creation of genetically engineered animal models for research.