A CRISPR–Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis

Abstract

Genetic manipulation is among the most important tools for synthetic biology; however, modifying multiple genes is extremely time‐consuming and can sometimes be impossible when dealing with gene families. Here, we present a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR‐associated protein 9 (Cas9) system for use in the diploid yeast Candida tropicalis that is vastly superior to traditional techniques. This system enables the rapid and reliable introduction of multiple genetic deletions or mutations, as well as a stable expression using an integrated CRISPR–Cas9 cassette or a transient CRISPR–Cas9 cassette, together with a short donor DNA. We further show that the system can be used to promote the in vivo assembly of multiple DNA fragments and their stable integration into a target locus (or loci) in C. tropicalis. Based on this system, we present a platform for the biosynthesis of β‐carotene and its derivatives. These results enable the practical application of C. tropicalis and the application of the system to other organisms.

Optimization of CRISPR‐Cas9 through promoter replacement and efficient production of L‐homoserine in Corynebacterium glutamicum

“…[ 28 ] The integrated expression of recET can enhance the HR ability of C. glutamicum and increase the efficiency of genome editing. Previous studies have also shown that the low level of the cas9 gene is adequate for genome editing, [ 29 ] and the heterologous expression of recombinant enzymes such as recET is very important for enhancing the HR ability of target strains. [ 30 ] In addition to Cas9, the high expression of sgRNA is also important for CRISPR‐Cas9 system.…”

Genetically Modified Yeasts in Wine Biotechnology

“…Komagataella phaffii), CRISPR-Cas9 system has been applied to improve its efficiency for the production off high-value pharmaceuticals [88]; in Ogataea polymorpha, a thermotolerant methylotrophic yeast, for the production of bioethanol [89] or for the introduction of all the genes necessary for the biosynthesis of resveratrol [90]. For biofuels and chemicals production in Issatchenkia orientalis [91]; in Kluyveromyces marxianus for its use as cell factory [92,93]; in Kluyveromyces marxianus for the production of recombinant proteins [94,95], or for integrating a synthetic muconic acid pathway [96]; in Schizosaccharomyces pombe [97]; in Candida species for the production of xylonic acid and ethanol [98] or for biosynthesis of β-carotene and its derivatives [99]; and in Yarrowia lipolytica for the production of renewable chemicals and enzymes for fuel, feed, oleochemical, nutraceutical and pharmaceutical applications [100].…”

CRISPR-Based Genetic Manipulation of Candida Species: Historical Perspectives and Current Approaches

“…Both an integrative and alternative transient CRISPR-Cas9 system were developed concurrently for use in C. tropicalis (Zhang et al, 2020 ). A number of candidate promoters were screened for efficient activity from the C. tropicalis genome, of which the GAP1 and/or FBA1 promoters were selected to express CAS9 and the sgRNA.…”

“…A number of candidate promoters were screened for efficient activity from the C. tropicalis genome, of which the GAP1 and/or FBA1 promoters were selected to express CAS9 and the sgRNA. Since pGAP1 and pFBA1 are RNA polymerase II promoters, the HH and HDV ribozyme sequences flanked the sgRNA (Zhang et al, 2020 ). Fusion of the DNA repair templates to the CRISPR-Cas9 construct resulted in homozygous single gene mutants at an efficiency of 83–100% when targeting the reporter genes ADE2 and URA3 , and up to 32% for the respective double mutant.…”

“…Fusion of the DNA repair templates to the CRISPR-Cas9 construct resulted in homozygous single gene mutants at an efficiency of 83–100% when targeting the reporter genes ADE2 and URA3 , and up to 32% for the respective double mutant. Like the first plasmid-based CRISPR-Cas9 system for C. tropicalis , this transient system rarely induces NHEJ events in the presence of a repair template and does not require integration of a selection marker into the genome (Zhang et al, 2020 ). Therefore, both CRISPR-Cas9 systems developed for C. tropicalis can successfully edit the genome at high efficiencies.…”