A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae.
Yueping ZhangJuan WangZibai WangYiming ZhangShuobo ShiJens B NielsenZihe LiuPublished in: Nature communications (2019)
With rapid progress in DNA synthesis and sequencing, strain engineering starts to be the rate-limiting step in synthetic biology. Here, we report a gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) for multiplexed engineering of Saccharomyces cerevisiae. Using reported gRNAs shown to be effective, this system enables simultaneous disruption of 8 genes with 87% efficiency. We further report an accelerated Lightning GTR-CRISPR that avoids the cloning step in Escherichia coli by directly transforming the Golden Gate reaction mix to yeast. This approach enables disruption of 6 genes in 3 days with 60% efficiency using reported gRNAs and 23% using un-optimized gRNAs. Moreover, we applied the Lightning GTR-CRISPR to simplify yeast lipid networks, resulting in a 30-fold increase in free fatty acid production in 10 days using just two-round deletions of eight previously identified genes. The GTR-CRISPR should be an invaluable addition to the toolbox of synthetic biology and automation.
Keyphrases
- saccharomyces cerevisiae
- genome editing
- crispr cas
- genome wide
- fatty acid
- escherichia coli
- single cell
- bioinformatics analysis
- genome wide identification
- high throughput
- high resolution
- dna methylation
- loop mediated isothermal amplification
- single molecule
- pseudomonas aeruginosa
- transcription factor
- circulating tumor
- staphylococcus aureus
- cystic fibrosis
- mass spectrometry
- klebsiella pneumoniae