Self-cloning CRISPR/Cpf1 facilitated genome editing in Saccharomyces cerevisiae

Abstract

Background: Saccharomyces cerevisiae is one of the most important industrial microorganisms. A robust genome editing tool is vital for both fundamental research and applications. To save the time and labor consumed in the procedure of genome editing, a self-cloning CRISPR/Cpf1 system (scCRISPR/Cpf1), in which a self-cleaving plasmid and PCR-generated site-specific crRNA fragment were included, was developed. Results: Using scCRISPR/Cpf1 as the genetic tool, simple and fast singleplex and multiplex genomic integration of in vivo assembled DNA parts were investigated. Moreover, we validate the applicability of scCRISPR/Cpf1 for cell factory development by creating a patchoulol production strain through two rounds of iterative genomic integration. The results showed that scCRISPR/Cpf1 enables singleplex and tripleplex genomic integration of in vivo assembled DNA parts with efficiencies of 80 and 32%, respectively. Furthermore, the patchoulol production strain was successfully and rapidly engineered and optimized through two rounds of iterative genomic integration by scCRISPR/Cpf1. Conclusions: scCRISPR/Cpf1 allows for CRISPR/Cpf1-facilitated genome editing by circumventing the step to clone a site-specific crRNA plasmid without compromising efficiency in S. cerevisiae. This method enriches the current set of tools available for strain engineering in S. cerevisiae.

CRISPR-Cas9-mediated Large Cluster Deletion and Multiplex Genome Editing in Paenibacillus polymyxa

“…The differences in regard of expression of the chromosomally integrated mRFP and sfGFP genes will need further investigations but can be linked to differences in the necessary promoter and RBS sequences. Furthermore, as the conjugation efficiency obtained from multiplexing approaches was rather low, optimization of transformation procedure might improve the efficiency for multiplex integration. , Previous studies have reported CRISPR-Cas-based multiplex genetic engineering in different microorganisms. − However, only a few demonstrated multiplex gene integration, which was mainly performed in yeast. , While it has been reported in E. coli, to the best of our knowledge, our study is the first to utilize a Cas9-based system to achieve multiplex gene integrations in Gram-positive bacteria.…”

CRISPR-Cas9-mediated Large Cluster Deletion and Multiplex Genome Editing in Paenibacillus polymyxa

“…[24][25][26] However, only a few successfully demonstrated multiplex gene integrations, which was mainly performed in yeast. 27,28 While multiplex gene insertion has been reported in E. coli, 29 to the best of our knowledge, our study is the first to utilize a Cas9-based system to achieve multiplex gene integrations in Gram-positive bacteria.…”

Tips, Tricks, and Potential Pitfalls of CRISPR Genome Editing in Saccharomyces cerevisiae

“…One important Cas protein that has been employed for genome editing is the Cas12a enzyme (also known as Cpf1), which recognizes a T-rich PAM motif ( Nakade et al, 2017 ; Yan et al, 2019 ; Paul and Montoya, 2020 ; Thompson et al, 2021 ) ( Table 2 ). Genome editing using Cas12a has a number of differences from Cas9 for genome editing in yeast: first, it does not require tracrRNA, therefore their sgRNAs are shorter relative to Cas9; second, it can process multiple crRNAs from a single crRNA array, whereas for Cas9 one must express crRNAs separately or process gRNAs using additional enzymes; third, its PAM motif is located on the 5’ end of the target DNA sequence whereas this is on the 3’ end for Cas9; and finally Cas12a generates staggered or sticky DSBs containing overhangs, often leading to deletions and point mutations ( Świat et al, 2017 ; Zetsche et al, 2017 ; Li Z.-H. et al, 2018 ; Verwaal et al, 2018 ; Ciurkot et al, 2019 ; Ciurkot et al, 2021 ). These properties of Cas12a make multiplexing experiments relatively simple relative to those for Cas9 which require multiple or even complex expression constructs.…”