Redirecting Metabolic Flux in E. Coli via Combinatorial CRISPRi-Mediated Repression for Isopentenol Production

The Science

Use CRISPR interference (CRISPRi) to knock down endogenous genes in competing pathways to redirect metabolic flux toward target metabolite.

The Impact

  • Designed a single-gRNA library with 15 individual knockdown targets, where 3 gRNA cassettes targeting genes asnA, prpE, and gldA increased isopentenol titer by 18% to 24%.
  • Combined the three single-gRNA cassettes into a two- or three-gRNA array and observed up to 98% enhancement in production by fine-tuning the repression level through titrating dCas9 expression.
  • Strategy shows that multiplex combinatorial knockdown of competing genes using CRISPRi can increase production of the target metabolite.


CRISPRi-mediated multiplex repression system to silence transcription of several endogenous genes to increase precursor availability in a heterologous isopentenol biosynthesis pathway.

Principal Investigator(s)

Taek Soon Lee
Lawrence Berkeley National Laboratory