Quantitative and modularized CRISPR/dCas9-dCpf1 dual function system in Saccharomyces cerevisiae

Frontiers in Bioengineering and Biotechnology

Bioengineering and Biotechnology

Chun Li¹  Lei Qin¹  Xiaoyu Ning²  Jun Li²  Qing Feng² 

[1]Key Lab for Industrial Biocatalysis, Department of Chemical Engineering, Ministry of Education, Tsinghua University, Beijing, China

[2]Key Laboratory of Medical Molecule Science and Pharmaceutics Engineering, School of Chemistry and Chemical Engineering, Ministry of Industry and Information Technology, Beijing Institute of Technology, Beijing, China

关键词: Saccharomyces cerevisiae;    CRISPR/dCas9-dCpf1;    bifunctional system;    quantitative;    modular;    β-carotene;   

DOI  :  10.3389/fbioe.2023.1218832

 received in 2023-05-08, accepted in 2023-10-05,  发布年份 2023

来源: Frontiers

【 摘 要 】

Introduction: Both CRISPR/dCas9 and CRISPR/dCpf1 genome editing systems have shown exciting promises in modulating yeast cell metabolic pathways. However, each system has its deficiencies to overcome. In this study, to achieve a compensatory effect, we successfully constructed a dual functional CRISPR activation/inhibition (CRISPRa/i) system based on Sp-dCas9 and Fn-dCpf1 proteins, along with their corresponding complementary RNAs.Methods: We validated the high orthogonality and precise quantity targeting of selected yeast promoters. Various activating effector proteins (VP64, p65, Rta, and VP64-p65-Rta) and inhibiting effector proteins (KRAB, MeCP2, and KRAB-MeCP2), along with RNA scaffolds of MS2, PP7 and crRNA arrays were implemented in different combinations to investigate quantitative promoter strength. In the CRISPR/dCas9 system, the regulation rate ranged from 81.9% suppression to 627% activation in the mCherry gene reporter system. Studies on crRNA point mutations and crRNA arrays were conducted in the CRISPR/dCpf1 system, with the highest transcriptional inhibitory rate reaching up to 530% higher than the control. Furthermore, the orthogonal CRISPR/dCas9-dCpf1 inhibition system displayed distinct dual functions, simultaneously regulating the mCherry gene by dCas9/gRNA (54.6% efficiency) and eGFP gene by dCpf1/crRNA (62.4% efficiency) without signal crosstalk.Results and discussion: Finally, we established an engineered yeast cell factory for β-carotene production using the CRISPR/dCas9-dCpf1 bifunctional system to achieve targeted modulation of both heterologous and endogenous metabolic pathways in Saccharomyces cerevisiae. The system includes an activation module of CRISPRa/dCas9 corresponding to a gRNA-protein complex library of 136 plasmids, and an inhibition module of CRISPRi/dCpf1 corresponding to a small crRNA array library. Results show that this CRISPR/dCas9-dCpf1 bifunctional orthogonal system is more quantitatively effective and expandable for simultaneous CRISPRa/i network control compared to single-guide edition, demonstrating higher potential of future application in yeast biotechnology.

【 授权许可】

Unknown   
Copyright © 2023 Feng, Ning, Qin, Li and Li.

【 图 表 】

fchem-11-1258026-fx7.tif

FBIOE_fbioe-2023-1218832_wc_tfx3.tif

FBIOE_fbioe-2023-1218832_wc_tfx2.tif

fneur-14-1255526-i002.tif