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

Qing Feng; Xiaoyu Ning; Lei Qin; Jun Li; Chun Li

doi:10.3389/fbioe.2023.1218832

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

Front Bioeng Biotechnol. 2023 Oct 18:11:1218832. doi: 10.3389/fbioe.2023.1218832. eCollection 2023.

Authors

Qing Feng¹, Xiaoyu Ning¹, Lei Qin², Jun Li¹, Chun Li²

Affiliations

¹ 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.
² Key Lab for Industrial Biocatalysis, Department of Chemical Engineering, Ministry of Education, Tsinghua University, Beijing, China.

Abstract

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.

Keywords: CRISPR/dCas9-dCpf1; Saccharomyces cerevisiae; bifunctional system; modular; quantitative; β-carotene.

Grants and funding

This work was supported by the National Natural Science Foundation of China under Grant No. 22078021, and National Key Research and Development Program of China under Grant 2022YFA0911803-04.