Development and characterization of a CRISPR/Cas9n-based multiplex genome editing system for .

BACKGROUND

Metabolic engineering has expanded from a focus on designs requiring a small number of genetic modifications to increasingly complex designs driven by advances in multiplex genome editing technologies. However, simultaneously modulating multiple genes on the chromosome remains challenging in . Thus, developing an efficient and convenient method for multiplex genome editing is imperative.

RESULTS

Here, we developed a CRISPR/Cas9n-based multiplex genome editing system for iterative genome editing in . This system enabled us to introduce various types of genomic modifications with more satisfying efficiency than using CRISPR/Cas9, especially in multiplex gene editing. Our system achieved at least 80% efficiency for 1-8 kb gene deletions, at least 90% efficiency for 1-2 kb gene insertions, near 100% efficiency for site-directed mutagenesis, 23.6% efficiency for large DNA fragment deletion and near 50% efficiency for three simultaneous point mutations. The efficiency for multiplex gene editing was further improved by regulating the nick repair mechanism mediated by gene, which finally led to roughly 65% efficiency for introducing three point mutations on the chromosome. To demonstrate its potential, we applied our system to simultaneously fine-tune three genes in the riboflavin operon and significantly improved the production of riboflavin in a single cycle.

CONCLUSIONS

We present not only the iterative CRISPR/Cas9n system for but also the highest efficiency for simultaneous modulation of multiple genes on the chromosome in reported to date. We anticipate this CRISPR/Cas9n mediated system to greatly enhance the optimization of diverse biological systems via metabolic engineering and synthetic biology.