Abstract
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.
Highlights
With rapid progress in DNA synthesis and sequencing, strain engineering starts to be the rate-limiting step in synthetic biology
The reasons for choosing tRNAGly are (i) tRNAGly has been applied in guide RNA (gRNA) processing in plants and Drosophila[9,10,11] and (ii) tRNAGlygene is relatively short with 71 base pairs compared to other endogenous tRNAs, and this enables a simple and compact architecture, so that gRNAs may be transcribed more efficiently
Mode A was a single transcript of gRNA-tRNAGly array (GTR) under a widely used RNA polymerase III promoter, SNR52 promoter[5,18]; mode B contained multiple gRNA expression cassettes with each cassette composed of one SNR52 promoter, one gRNA, and one SNR52 terminator; and mode C contained multiple gRNA expression cassettes, and from the second cassette on, each cassette was composed of one fusion promoter of SNR52 promoter and tRNAGly sequence, one gRNA, and one SNR52 terminator
Summary
With rapid progress in DNA synthesis and sequencing, strain engineering starts to be the rate-limiting step in synthetic biology. 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. 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. For un-optimized gRNAs, the Lightning GTR-CRISPR enabled 6-HIS-gene disruptions at 23% efficiency in 3 days This system greatly accelerates the speed of yeast strain development, and we achieved an increase in free fatty acid (FFA) production by about 30-fold in 10 days using just 2-round deletions of 8 previously identified genes
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