NLS-Fusions to Cas9 Can Improve Editing

Daniel Hasegawa, Ph.D is a Research Molecular Biologist with the USDA -U.S. Vegetable Laboratory, in Savannah, South Carolina and is interested in understanding the molecular and physiological processes that drive insect-virus relationships. MORE ABOUT THE AUTHOR

In a recent study using zebrafish published in , Hu et al. (2018) conducted a comprehensive analysis of gene editing efficiency between Cas9 proteins and Cas9 mRNA, each fused with a nuclear-localization signal (NLS) in a different position. Overall, five Cas9 variants were compared:

  • N-terminal NLS-Cas9 protein
  • C-terminal NLS-Cas9 protein
  • N- and C-terminal NLS Cas9 protein
  • Cas9 protein without NLS
  • N- and C-terminal Cas9 mRNA

Two genes related to body pigmentation were targeted by microinjecting zebrafish embryos with gRNA and one of the Cas9 variants. Two gRNAs were selected for each gene, and multiple pieces of data were collected to assess gene editing efficiency, including: mutation rate, indel frequency, and type of indel. In addition, phenotypic data was collected to relate gene editing efficiency to function.

When the authors performed single target knockouts, high levels of gene editing efficiency and phenotypic mutation was observed for all three NLS-fused Cas9 proteins and the N-Cas9-C mRNA, (although the efficiency did vary between gRNAs).

File:Rancycle nuclearimport nuclearexport.png

Proteins, are actively transported across the nuclear membrane in a process called the Ran-GTP nuclear transport cycle.

Interestingly, the Cas9 protein without an NLS was shown to generate mutation rates comparable to the Cas9 variants with an NLS at one of the target sites. These data suggest that the position of the NLS on Cas9 protein and mRNA does not affect the gene editing efficiency of the CRISPR/Cas9 system in zebrafish and that in some cases, and NLS may not be necessary for Cas9 nuclear import. The exact mechanism as to how this occurs would make for an interesting follow-up study.

Cas9 with functional domains highlighted in relation to target DNA and guideRNA

Next, Hu et al., explored the potential of the Cas9 variants to be used in multiplex genome editing. After injecting zebrafish embryos with each of the five Cas9 variants simultaneously with all four gRNAs (two genes, each with two sites), the authors found that the efficiencies of multiplex genome editing were similar to the efficiencies when targeting single sites. Again, the mutagenesis rate of the Cas9 protein without an NLS varied between sites.

Overall, Hu et al., provides a thorough assessment of gene editing efficiency by Cas9 proteins and mRNA, and conclude that high levels of mutagenesis can be achieved regardless of the position of the fused NLS. These data will help researchers during the selection process of Cas9 enzymes to use for their own studies, and may be particularly beneficial for those that are looking to tag their Cas9 enzyme for visualization and detection purposes.

Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish
Peinan Hu, Xueying Zhao, Qinghua Zhang, Weiming Li and Yao Zu
G3: Genes, Genomes, Genetics March 1, 2018 vol. 8 no. 3 823-831; DOI; 10.1534/g3.117.300359



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