Abstract
Objectives
To test the applicability of Cpf1 from Francisella novivida in genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae.
Results
An easy-to-use vector toolkit, containing a CEN6/ARS4 plasmid expressing Cpf1 from Francisella novivida (FnCpf1) and a 2 μ plasmid for crRNA or crRNA array expressing, was constructed for Cpf1-assisted genomic integration in S. cerevisiae. Our results showed that FnCpf1 allowed for targeted singleplex, doubleplex, and tripleplex genomic integration of in vivo assembled DNA parts with efficiencies of 95, 52, and 43%, respectively.
Conclusions
CRISPR-Cpf1 system allows for efficient genomic integration of in vivo assembled DNA parts in S. cerevisiae, and thus provides an alternative CRISPR-Cas method for metabolic pathway engineering in addition to CRISPR-Cas9 system previously reported for yeast.
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Acknowledgements
This research was financially supported by the National Natural Science Foundation of China (No. 31500043). The authors sincerely thank Prof. Hongwei Yu (Zhejiang University) for providing plasmids of pESC-LEU-cen, pESC-URA, pMRI-34-crtE-tHMG1, pMRI-35-crtYB-crtI, pUMRI-15, and strain of S. cerevisiae BY4741.
Supporting information
Supplementary Fig. 1—Composition of the CRISPR arrays for singleplex, doubleplex, and tripleplex genome editing target sites.
Supplementary Fig. 2—Graphical depiction of the positions of the genomic target sites of the Gal1-7, Gal80, and HO for FnCpf1.
Supplementary Fig. 3—Pictures of transformants to determine the efficiency of targeted singleplex, doubleplex, and tripleplex genomic integration facilitated by FnCpf1. A: Pictures of transformants obtained by co-transforming donor DNA of MEL1 with plasmid expressing singleplex crRNA (Gal1-7_pos1, Gal1-7_pos2, and Gal1-7_pos3) or without crRNA plasmid (control1). B: Pictures of transformants obtained by co-transforming donor DNA of MEL1 and KanMX with plasmid expressing doubleplex crRNA array (Gal1-7_pos2 + Gal80_pos1, Gal1-7_pos2 + Gal80_pos2, and Gal1-7_pos2 + Gal-80_pos3) or without crRNA plasmid (control2). C: Pictures of transformants obtained by co-transforming donor DNA of crtI, crtYB, and tHMG1-crtE with plasmid expressing tripleplex crRNA array (Gal1-7_pos2 + Gal80_pos1 + HO_pos1, Gal1-7_pos2 + Gal80_pos1 + HO_pos2, and Gal1-7_pos2 + Gal80_pos1 + HO_pos3) or without crRNA plasmid (control3).
Supplementary Table 1—Primers and oligonucleotides used in this study.
Supplementary Table 2—Donor DNA flank and expression cassette sequences.
Supplementary Table 3—Synthesized DNA fragments in this study.
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Li, ZH., Liu, M., Wang, FQ. et al. Cpf1-assisted efficient genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae. Biotechnol Lett 40, 1253–1261 (2018). https://doi.org/10.1007/s10529-018-2574-8
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DOI: https://doi.org/10.1007/s10529-018-2574-8