Guide RNA Engineering Enables Dual Purpose CRISPR-Cpf1 for Simultaneous Gene Editing and Gene Regulation in <i>Yarrowia lipolytica</i>
Adithya Ramesh, Thomas Prates Ong, Jaime Garcia, Jessica R. Adams, Ian Wheeldon
Abstract
has fast become a biotechnologically significant yeast for its ability to accumulate lipids to high levels. While there exists a suite of synthetic biology tools for genetic engineering in this yeast, there is a need for multipurposed tools for rapid strain generation. Here, we describe a dual purpose CRISPR-Cpf1 system that is capable of simultaneous gene disruption and gene regulation. Truncating guide RNA spacer length to 16 nt inhibited nuclease activity but not binding to the target loci, enabling gene activation and repression with Cpf1-fused transcriptional regulators. Gene repression was demonstrated using a Cpf1-Mxi1 fusion achieving a 7-fold reduction in mRNA, while CRISPR-activation with Cpf1-VPR increased hrGFP expression by 10-fold. High efficiency disruptions were achieved with gRNAs 23-25 bp in length, and efficiency and repression levels were maintained with multiplexed expression of truncated and full-length gRNAs. The developed CRISPR-Cpf1 system should prove useful in metabolic engineering, genome wide screening, and functional genomics studies.