A CRISPR/Cas9-Mediated, Homology-Independent Tool Developed for Targeted Genome Integration in Yarrowia lipolytica

Abstract

Yarrowia lipolytica has been extensively used for producing essential chemicals and enzymes. Like most other eukaryotes, non-homologous end joining (NHEJ) is the major repair pathway for DNA double strand breaks in Y. lipolytica. Although numerous studies have attempted to achieve targeted genome integration through homologous recombination (HR), this process requires the construction of homologous arms, which is time-consuming. This study aimed to develop a homology-independent and CRISPR/Cas9-mediated targeted genome integration tool in Y. lipolytica. Through optimizing the cleavage efficiency of Cas9, targeted integration of a hyg fragment was achieved with12.9% efficiency, which was further improved by manipulation of the fidelity of NHEJ repair, cell cycle, and the integration sites. Thus, targeted integration rate reached to 55% through G1 phase synchronization. This tool was successfully applied for the rapid verification of intronic promoters and iterative integration of four genes pathway for canthaxanthin biosynthesis. This homology-independent integration tool does not require homologous templates and selection markers, and achieves one-step targeted genome integration of the 8417 bp DNA fragment, potentially replacing current HR-dependent genome editing methods for Y. lipolytica. IMPORTANCE This study describes the development and optimization of a homology-independent targeted genome integration tool mediated by CRISPR/Cas9 in Yarrowia lipolytica. This tool does not require the construction of homologous templates, and can be used to rapidly verify genetic elements and iteratively integrate of multiple genes pathway in Y. lipolytica. This tool may serve as a potential supplement for current HR-dependent genome editing methods for eukaryotes.

YALIcloneNHEJ: An Efficient Modular Cloning Toolkit for NHEJ Integration of Multigene Pathway and Terpenoid Production in Yarrowia lipolytica

“…Preparing the Modular Parts for Golden Gate Assembly NHEJ was recently found to be a promising method for chromosomal integration in Y. lipolytica (Cui et al, 2021). However, there is currently no modular assembly tool for the construction of multi-gene biosynthetic pathways applied for NHEJ integration, which makes genetic manipulation timeconsuming and laborious.…”

“…In comparison with homologous recombination, NHEJ has many unique advantages, including high integration efficiency and no need for a homologous template ( Liu et al, 2019 ). Moreover, the heterologous DNA can be randomly inserted into the chromosome, which results in a library of multilocus integrants that can facilitate the screening of hyperproducer strains ( Ye et al, 2012 ; Friedlander et al, 2016 ; Cui et al, 2021 ). However, there was no multigene plasmid construction method for NHEJ-based integration, which made it challenging to construct and integrate metabolic pathways in Y. lipolytica .…”

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

“…The targeted gene integration rate was up to 55% by optimizing the cleavage efficiency of Cas9, manipulating repair fidelity of NHEJ, cell cycle and integration sites. By using this tool, iterative integration of canthaxanthin biosynthesis pathway including four genes ( GGS1 , carB , carRP and CrtW ) was achieved ( Cui et al, 2021 ). It is worth noting that integration of an 8,417 bp fragment composed of GGS1 , carB , and carRP into genome by one step may still be a challenge for the HR dependent targeted genome integration, indicating this tool paves a new avenue to realize the accurate and efficient targeted genome integration in some non-conventional yeasts.…”

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

“…Briefly, from 2016, several efficient CRISPR–Cas9 systems for single and multigene editing as well repression or activation of genes in Y. lipolytica are being developed (Gao et al, 2016; C. Schwartz et al, 2017, 2018; C. M. Schwartz et al, 2016; Z. Yang et al, 2020). 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).…”