Optimizing Gene Drive Performance

Justin Overcash is a working on his Ph.D. in Dr. Zach Adelman’s laboratory at Texas A & M University More About the Author

Roggenkamp et al 2018, in a recent publication in G3:Genes, Genomes, Genetics provide an in-depth analysis of potential ways that gene drive mechanisms can be regulated by targeting four conserved elements which would be found in any CRISPR/Cas9 based gene drive system.

Utilizing a yeast-based system, Roggenkamp et al addressed conserved elements including inducible promoters, sgRNA length/identity, nuclear localization of Cas9 and Cas9 fusion proteins.  The lessons learned from this system should be portable to other eukaryotes in which synthetic gene drives are being introduced, according to the authors.

The genetics of gene drive illustrating the transmission pattern of a ‘selfish’ gene (red).  Synthetic biologists can now assemble and introduce genetic elements with these properties into organisms of their choice.

The ability to modulate gene drives and the identification of extra layers of control begins to address some of the safety and ethical questions associated with their use.

Roggenkamp et al first assessed the use of an inducible promoter (GAL1/10) showing reliable use in producing indels and in a gene drive system. Most notably it was observed that drive efficiency increases the longer Cas9 is expressed, with full drive efficiency at eight to twelve hours.

Secondly, sgRNA length was assessed for efficiency of both indel production and gene drive efficiency. Varied length of sgRNAs (16-22bp) and mismatches were assessed showing that a 19-22bp guide is required and with one exception (19bp guide with 1bp mismatch) sgRNAs either worked completely or did not work at all.

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

One of the more interesting assessments was the use of nuclear localization (NLS) and export (NES) signals and their combined use to modulate gene drive efficiency. Utilizing Cas9-EGFP fusion proteins with a varied number of nuclear localization/export signals or a combination of both lead to a range of gene drive efficiencies.

Lastly, Cas9 fusion proteins with Cas9 being fused to a “dead” Cas9 variant (dCas9) were assessed showing the utilization of Cas9-dCas9 can be utilized to lower gene drive efficiency.

Image result for nuclear localization signal

Generalized scheme for the importation of proteins containing nuclear localization signals. Driving Cas9 into the nucleus with a NLS was found to increase endonuclease activity.   Image Credits https://www.proprofs.com/flashcards/story.php?title=mcb-block-3-organelles

Roggenkamp et al 2018 show that multiple elements within a Cas9 gene drive system can be controlled by researchers to modulate gene drive efficiency in their organism of choice. While factors such as DNA repair, identification of inducible promoters and nuclear localization will need to be addressed in any organism in which a drive system is utilized, the experiments conducted provide interesting insights into variables which should be considered when tailoring a gene drive system.

Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae Emily Roggenkamp,Rachael M. Giersch,Madison N. Schrock,Emily Turnquist,Megan Halloran,Gregory C. FinniganG3: Genes, Genomes, Genetics March 1, 2018 vol. 8 no. 3 999-1018; https://doi.org/10.1534/g3.117.300557

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