Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System
- Authors
- Hwang, W.Y., Fu, Y., Reyon, D., Maeder, M.L., Kaini, P., Sander, J.D., Joung, J.K., Peterson, R.T., and Yeh, J.R.
- ID
- ZDB-PUB-130805-29
- Date
- 2013
- Source
- PLoS One 8(7): e68708 (Journal)
- Registered Authors
- Peterson, Randall, Yeh, Jing-Ruey (Joanna)
- Keywords
- none
- 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*
- PubMed
- 23874735 Full text @ PLoS One
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.