Guide RNA Engineering Enables Dual Purpose CRISPR-Cpf1 for Simultaneous Gene Editing and Gene Regulation in Yarrowia lipolytica

• DOI: 10.1021/acssynbio.9b00498
• Publication Date: 2020-03-25T20:58:17Z
• Authors: Adithya Ramesh; Thomas Ong; Jaime A. Garcia; Jessica Adams; Ian Wheeldon
• Keywords: Yarrowia; Synthetic Biology; Genome Engineering; Guide RNA; Trans-activating crRNA; Functional Genomics; CRISPR interference; Metabolic Engineering; Zinc finger nuclease

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