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

Heritable and precise zebrafish genome editing using a CRISPR-Cas system.

Author information

1
Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America.

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.

PMID:
23874735
PMCID:
PMC3706373
DOI:
10.1371/journal.pone.0068708
[Indexed for MEDLINE]
Free PMC Article

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