GTR 2.0: gRNA-tRNA Array and Cas9-NG Based Genome Disruption and Single-Nucleotide Conversion in Saccharomyces cerevisiae

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 gRNAtRNA 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.

Automated summary

This study developed an efficient tool for genome disruption and single-nucleotide conversion, utilizing a gRNAtRNA array and SpCas9-NG (GTR 2.0). The tool achieved near 100% efficiency for gene disruptions in yeast cells covering all 16 possible NGN PAMs and all 12 possible single-nucleotide conversions (N to N). Furthermore, the tool was able to achieve 66.67% mutation efficiency for multiplexed single-nucleotide conversions and 100% mutation efficiency for generating multiple conversions in different genes simultaneously. GTR 2.0 will significantly enhance the scope, efficiency, and capabilities of yeast genome editing, serving as a versatile and valuable addition to the toolbox of synthetic biology and metabolic engineering.