PUBLICATION
Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide RNAs
- Authors
- Gagnon, J.A., Valen, E., Thyme, S.B., Huang, P., Ahkmetova, L., Pauli, A., Montague, T.G., Zimmerman, S., Richter, C., Schier, A.F.
- ID
- ZDB-PUB-140531-13
- Date
- 2014
- Source
- PLoS One 9: e98186 (Journal)
- Registered Authors
- Huang, Peng, Pauli, Andrea, Schier, Alexander, Thyme, Summer, Zimmerman, Steve
- Keywords
- none
- MeSH Terms
-
- Alleles
- Animals
- Gene Frequency
- Humans
- INDEL Mutation
- Mutagenesis*
- Mutation Rate
- Oligonucleotides/genetics*
- RNA, Guide, Kinetoplastida/genetics*
- RNA, Guide, Kinetoplastida/metabolism
- Zebrafish/genetics
- Zebrafish/metabolism
- PubMed
- 24873830 Full text @ PLoS One
Citation
Gagnon, J.A., Valen, E., Thyme, S.B., Huang, P., Ahkmetova, L., Pauli, A., Montague, T.G., Zimmerman, S., Richter, C., Schier, A.F. (2014) Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide RNAs. PLoS One. 9:e98186.
Abstract
The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping