A Novel and Efficient Genome Editing Tool Assisted by CRISPR-Cas12a/Cpf1 for Pichia pastoris

Abstract

Pichia pastoris has been widely exploited for the heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value chemicals and pharmaceuticals. Effective synthetic biology tools for genetic engineering are essential for industrial and biotechnological research in this yeast. Here, we describe a novel and efficient genome editing method mediated by the CRISPR-Cpf1 system, which could facilitate the deletion of large DNA fragments and integration of multiplexed gene fragments. The CRISPR-Cpf1 system exhibited a precise and high editing efficiency for single-gene disruption (99 ± 0.8%), duplex genome editing (65 ± 2.5% to 80 ± 3%), and triplex genome editing (30 ± 2.5%). In addition, the deletion of large DNA fragments of 20kb and one-step integration of multiple genes were first achieved using the developed CRISPR-Cpf1 system. Taken together, this study provides an efficient and simple gene editing tool for P. pastoris. The novel multiloci gene integration method mediated by CRISPR-Cpf1 may accelerate the ability to engineer this methylotrophic yeast for metabolic engineering and genome evolution in both biotechnological and biomedical applications.

CRISPR/Cas9-Mediated Multiplexed Genome Editing in Aspergillus oryzae

“…Achieving simultaneous editing of multiple genes is beneficial to improve the use efficiency of selection markers and analyze the cooperation of many related genes [ 31 ]. Currently, the CRISPR/Cas system has progressed in multi-gene editing in filamentous fungi.…”

Overproduction of Patchoulol in Metabolically Engineered Komagataella phaffii

“…The Cre/loxP Zeo R marker recycling method and the CRISPR-Cpf1 systems were used to generate the recombinant yeast strain. , All yeast strains used in this study are listed in Table S1.…”

“…Pichia pastoris) is not only as a potential host for producing various high-value biochemical products like Saccharomyces cerevisiae , especially for its advantages in efficiently expressing heterologous enzymes to high levels, particularly those with plant or fungal origins that cannot be compatibly expressed in bacteria, and but also easy to grow to high cell densities and achieve scale-up cultivation in simple and inexpensive media. Consequently, it has been employed as a potential microbial cell factory to produce various highly valued compounds. − More specifically, many terpenoids, including carotenoids, (+)-nootkatone, dammarenediol-II, , lycopene, , α-farnesene, and α-santalene, , were produced by engineered K. phaffii.…”

“…Consequently, it has been employed as a potential microbial cell factory to produce various highly valued compounds. 3−6 More specifically, many terpenoids, including carotenoids, 7 (+)-nootkatone, 8 dammarenediol-II, 9,10 lycopene, 11,12 α-farnesene, and α-santalene, 13,14 were produced by engineered K. phaffii. However, there are no reports about the heterologous production of patchoulol by engineering K. phaffii with methanol as the sole carbon source.…”

High-Yield Natural Vanillin Production by Amycolatopsis sp. after CRISPR-Cas12a-Mediated Gene Deletion

“…The recent development of the CRISPR genome editing system was a major advance in genetic manipulation. , The CRISPR system is more efficient and faster than conventional homologous recombination editing because it can disrupt multiple genes simultaneously, greatly reducing the workload of gene editing. , The CRISPR/Cas9 system has been developed as a powerful genomic editing tool that has been used successfully in various species; − however, attempts to use it for genomic editing of Amycolatopsis spp. and some Streptomyces strains failed because of the high cytotoxicity of Cas9. , CRISPR-Cas12a is another CRISPR-Cas system with great potential for genome editing, which, unlike Cas9, can achieve pre-crRNA maturation and then target T-rich protospacer adjacent motifs (PAMs) under the guidance of single-stranded mature crRNAs to complete the cleavage of the target DNA .…”