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. In Press

Subject Categories

Medical Genetics

Bioinformatics and Systems Biology

Genetics

DOI

10.1021/acssynbio.0c00560

PubMed

34015926

More information

Latest update

6/29/2021