Dual CRISPR‐Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica

Abstract

CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica.

Homology‐independent genome integration enables rapid library construction for enzyme expression and pathway optimization in Yarrowia lipolytica

“…Using these tools, four genes of the lycopene biosynthesis pathway were integrated into Y. lipolytica at different locations (Schwartz et al, ). D. Gao, Smith, Spagnuolo, Rodriguez, and Blenner () demonstrated a homology‐mediated end joining‐based method for the integration of large DNA fragments that is twice as efficient as HR‐based methods. In addition, a method called DNA assembler, allowing assembly and integration of multiple fragments via in vivo HR, was reported for one‐step genome integration of three genes of the β‐carotene biosynthetic pathway in Y. lipolytica (S. Gao et al, ).…”

Advances and opportunities in gene editing and gene regulation technology for Yarrowia lipolytica

“…CRISPR–Cas9 systems often achieve gene knockouts via indels resulting in frameshift mutation, which can lead to the production of short nonsense proteins of unknown function. In an effort to alleviate potential problems caused by such proteins, Gao et al [56] demonstrates a CRISPR-based full gene excision knockout strategy. The system exploits Y. lipolytica ’s non-homologous end-joining (NHEJ) to make the repair of the two simultaneous DSBs.…”

A novel and effective Cre/lox-based genetic tool for repeated, targeted and markerless gene integration

“…A previous study examined 17 loci for targeted and markerless gene integration in Y. lipolytica , among which only five loci exhibited gene integration, with highest integration efficiency of 68.9%±25.5% (Schwartz et al, 2017b). Another study accomplished gene integration into PEX 10 locus via homologous recombination (HR) and homology-mediated end-joining, with integration efficiencies of 6.7%±3.6% and 37.5%±8.8%, respectively (Gao et al, 2018). These studies indicate the need for further improvement in achieving more desirable gene integration efficiency (similar to gene disruption efficiency of about 90%) via CRISPR-Cas9-based tools.…”

“…These studies indicate the need for further improvement in achieving more desirable gene integration efficiency (similar to gene disruption efficiency of about 90%) via CRISPR-Cas9-based tools. Besides, gene insertion via HR-mediated double-strand break (DSB) repair usually demands synchronous introduction of two plasmids harboring different selection markers (Gao et al, 2018; Schwartz et al, 2017b), resulting in reduction of transformation efficiency and additional procedure for plasmids recovery.…”