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

Abstract

Yarrowia lipolytica 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.

Advances and Opportunities of CRISPR/Cas Technology in Bioengineering Non-conventional Yeasts

“…Truncating sgRNA could inhibit the nuclease activity of Cas protein, but not influence the targeting effect. On this basis, a Cpf1-VPR fusion with truncated sgRNA (16 bp) increased hrGFP expression by 10-fold in Y. lipolytica ( Ramesh et al, 2020 ).…”

“…In C. glabrata , the choice of the promoter may influence the type of mutation, a single base pair or larger insertions were observed when different promoters were used to express Cas9 ( Enkler et al, 2016 ) . In Y. lipolytica , different promoters were screened for nCas9-pmCDA1-UGI expression, and the highest efficiency was achieved by TFFin promoter ( Ramesh et al, 2020 ). These findings show that an appropriate level of Cas9 expression is beneficial to the strain’s resilience and genome editing efficiency.…”

Systems‐level approaches for understanding and engineering of the oleaginous cell factory Yarrowia lipolytica

“…The gene integration efficiency of up to 55% has been achieved using a homology‐independent targeted genome integration tool mediated by CRISPR/Cas9 which does not require the construction of homologous templates (Cui et al, 2021). More recently, a dual‐purpose CRISPR‐Cpf1 system has been suggested that is capable of simultaneous gene disruption and gene regulation in Y. lipolytica (Ramesh et al, 2020). Furthermore, a suite of genetic tools (EasyCloneYALI) has been generated that facilitates transformation protocols by providing a series of pre‐designed plasmids and oligos (Holkenbrink et al, 2018).…”

Genome-wide functional screens enable the prediction of high activity CRISPR-Cas9 and -Cas12a guides in Yarrowia lipolytica

“…To create the LbCas12a sgRNA expression plasmid (pLbCas12ayl), we first added a second direct repeat sequence at the 5' of the polyT terminator in pCpf1_yl (see ref. 41 ). This was done to ensure that library sgRNAs could end in one or more thymine residues without being construed as part of the terminator.…”