Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System

PLoS One. 2013 Jul 9;8(7):e68708. doi: 10.1371/journal.pone.0068708. Print 2013.

Abstract

We have previously reported a simple and customizable CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided Cas9 nuclease (RGN) system that can be used to efficiently and robustly introduce somatic indel mutations in endogenous zebrafish genes. Here we demonstrate that RGN-induced mutations are heritable, with efficiencies of germline transmission reaching as high as 100%. In addition, we extend the power of the RGN system by showing that these nucleases can be used with single-stranded oligodeoxynucleotides (ssODNs) to create precise intended sequence modifications, including single nucleotide substitutions. Finally, we describe and validate simple strategies that improve the targeting range of RGNs from 1 in every 128 basepairs (bps) of random DNA sequence to 1 in every 8 bps. Together, these advances expand the utility of the CRISPR-Cas system in the zebrafish beyond somatic indel formation to heritable and precise genome modifications.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • CRISPR-Associated Proteins / chemistry
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Cas Systems*
  • Genetic Engineering
  • Genome*
  • Germ-Line Mutation
  • INDEL Mutation
  • Molecular Sequence Data
  • Mutagenesis, Insertional
  • Mutation Rate
  • Oligonucleotides / chemistry
  • Oligonucleotides / genetics
  • Sequence Alignment
  • Zebrafish / genetics*

Substances

  • CRISPR-Associated Proteins
  • Oligonucleotides