GTR 2.0: GRNA-tRNA Array and Cas9-NG Based Genome Disruption and Single-Nucleotide Conversion in Saccharomyces cerevisiae
Journal article, 2021
Targeted genome disruptions and single-nucleotide conversions with the CRISPR/Cas system have greatly facilitated the development of gene therapy, basic biological research, and synthetic biology. With vast progress in this field, there are still aspects to be optimized, including the target range, the ability to multiplex, the mutation efficiency and specificity, as well as the requirement of adjusting protospacer adjacent motifs (PAMs). Here, we report the development of a highly efficient genome disruption and single-nucleotide conversion tool with a gRNA-tRNA array and SpCas9-NG (GTR 2.0). We performed gene disruptions in yeast cells covering all 16 possible NGN PAMs and all 12 possible single-nucleotide conversions (N to N) with near 100% efficiencies. Moreover, we applied GTR 2.0 for multiplexed single-nucleotide conversions, resulting in 66.67% mutation efficiency in simultaneous generation of 4 single-nucleotide conversions in one gene, as well as 100% mutation efficiency for simultaneously generating 2 single-nucleotide conversions in two different genes. GTR 2.0 will substantially expand the scope, efficiency, and capabilities of yeast genome editing, and will be a versatile and invaluable addition to the toolbox of synthetic biology and metabolic engineering.
multiplex
Saccharomyces cerevisiae
SpCas9-NG
genome disruption
single-nucleotide conversion
Author
Guiping Gong
Beijing University of Chemical Technology
Yueping Zhang
China Agricultural University
Zibai Wang
Beijing University of Chemical Technology
Luo Liu
Beijing University of Chemical Technology
Shuobo Shi
Beijing University of Chemical Technology
Verena Siewers
BioInnovation Institute
Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology
Qipeng Yuan
Beijing University of Chemical Technology
Jens B Nielsen
Beijing University of Chemical Technology
Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology
BioInnovation Institute
Xu Zhang
Beijing University of Chemical Technology
Zihe Liu
Beijing University of Chemical Technology
ACS Synthetic Biology
2161-5063 (eISSN)
Vol. 10 6 1328-1337Subject Categories
Medical Genetics
Bioinformatics and Systems Biology
Genetics
DOI
10.1021/acssynbio.0c00560
PubMed
34015926