Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression

Abstract

SUMMARY Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…[144] Contrary to MAGE, CRISPR could be generally applied to many organisms such as bacteria, yeast, plants, and animals including humans. [143] In particular, transcriptional repression by CRISPR interference (CRISPRi), [145] illustrated in Figure 11b, has been applied to the production of several natural products. [146] In a study by Wu et al, multiple gene targets for increasing intracellular malonyl-CoA pool in the central metabolic pathways were downregulated by the CRISPRi system for naringenin production in E. coli.…”

“…b) CRISPRi system utilizes a single-guide RNA (sgRNA) and a dead Cas9 (dCas9) protein to block transcriptional initiation or elongation by binding to complementary DNA regions. [145,294] Thus, fine-tuning of target endogenous genes can be achieved. c) For screening high-performing mutants, QdoR or FdeR regulatory systems are used for flavonoid production.…”

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

“…using two adjacent gRNAs with Cas9n, can efficiently introduce both indel mutations and HR events with a single-stranded DNA oligo-nucleotide donor template in mammalian cells [28,10,80]. Complete disruption of the endonuclease activities (RuvC D10A along with HNH H840A ) results in a catalytically inactive Cas9, or dead-Cas9 (dCas9) [78,79]. This has been exploited to physically block the transcriptional machinery when targeted in the promoter region of a gene of interest, coined CRISPR interference (CRISPRi) [22,34] (Fig.…”

“…strong, weak, and inducible promoters [51]. As mentioned above, besides its efficient endonuclease activity, CRISPR can enable gene expression modulation through the deactivated form of the Cas9 protein, dCas9 [59,79]. Once bound to, or in the vicinity of the transcriptional start site (TSS), the gRNA:dCas9 complex can significantly alter the transcriptional expression by physically interfering with RNA polymerase binding [14,43,79].…”

“…As mentioned above, besides its efficient endonuclease activity, CRISPR can enable gene expression modulation through the deactivated form of the Cas9 protein, dCas9 [59,79]. Once bound to, or in the vicinity of the transcriptional start site (TSS), the gRNA:dCas9 complex can significantly alter the transcriptional expression by physically interfering with RNA polymerase binding [14,43,79]. Wu et al recently exploited this strategy in E. coli where they did a selective knockdown of gene expression of enzymes that could divert the carbon flux away from the production of 1,4-Butanediol (BDO) [99].…”