PUBLICATION

Genome Editing in Zebrafish Using High-Fidelity Cas9 Nucleases: Choosing the Right Nuclease for the Task

Authors
Prykhozhij, S.V., Cordeiro-Santanach, A., Caceres, L., Berman, J.N.
ID
ZDB-PUB-200202-3
Date
2020
Source
Methods in molecular biology (Clifton, N.J.)   2115: 385-405 (Chapter)
Registered Authors
Berman, Jason, Caceres, Lucia, Prykhozhij, Sergey
Keywords
CRISPR, Cas9, Genome editing, HiFi Cas9, HypaCas9, Knock-in, Mutation, Off-target, Zebrafish, sgRNA
MeSH Terms
  • Animals
  • CRISPR-Associated Protein 9/genetics*
  • CRISPR-Cas Systems*
  • Gene Editing/methods*
  • Microinjections
  • Mutagenesis, Site-Directed
  • RNA, Guide/genetics
  • Zebrafish/genetics*
PubMed
32006412 Full text @ Meth. Mol. Biol.
Abstract
Shortly after the development of the CRISPR/Cas9 system, it was recognized that it is prone to induce off-target mutations at significant frequencies. Therefore, there is a strong motivation to develop Cas9 enzymes with reduced off-target activity. Multiple rational design or selection approaches have been applied to develop several Cas9 versions with reduced off-target activities (high fidelity). To make these high-fidelity Cas9s available for model systems other than human cells and bacterial strains, as, for example, in zebrafish, new specialized expression vectors need to be developed. In this chapter, we focused on the HypaCas9 and HiFi Cas9 high-fidelity enzymes and incorporated the mutations of these Cas9 versions into a codon-optimized zebrafish Cas9 vector. This optimized vector was further improved by introducing an artificial polyadenine insert (A71) since polyadenylation is known to enhance mRNA translational efficiency. The Hypa-nCas9n and HiFi-nCas9n vectors were produced by single-site mutagenesis from pT3TS-nCas9n-A71 vector. We then tested the polyadenylated mRNAs for nCas9n, Hypa-nCas9n, HiFi-nCas9n, and HiFi-Cas9 protein for editing efficiency in five genome editing strategies and found that these high-fidelity Cas9 versions had different performances ranging from activity at 2-4 sites, where the wild-type nCas9n is active, indicating that these Cas9 versions have different sgRNA preferences. In summary, the developed new high-fidelity Cas9 vectors will enable researchers to perform much more accurate genome editing.
Genes / Markers
Figures
Show all Figures
Expression
Phenotype
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping
Errata and Notes