Nature biotechnologyJun 2, 2026

Controlling multiple bacterial genes independently using adjustable Cas13d systems

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jun 27, 2026

Abstract

Essence

Attenuated Cas13d variants may enable tunable, multiplexed RNA-level gene control in bacteria with reduced toxicity.

Evidence

This bacterial synthetic-biology platform experiment engineered Cas13d variants and tested transcript knockdown, translation inhibition, CRISPR activation, synthetic circuits, and lycopene pathway optimization in Escherichia coli.

Caveat

The work demonstrates toolkit performance in bacterial engineering systems, so its usefulness outside these tested contexts remains unproven.

Simplified

Cas13-based RNA effectors may enable dynamic, multiplexed and reversible gene regulation in bacteria. Yet, their widespread adoption is hindered by inherent cytotoxicity and collateral cleavage. Here we present a rational protein engineering strategy to generate attenuated Cas13d variants with tunable RNase activity through targeted truncation of flexible regions. This permits effective transcript knockdown while greatly reducing toxicity as reflected by a 2.2-fold higher growth optical density. By introducing proximal mismatches at the 5' end of CRISPR RNA spacers, our system allows functional switching between translation inhibition, polycistronic mRNA degradation and IF3-fusion-based translation-level CRISPR activation. We demonstrate programmable, orthogonal and multiplexed regulation of individual genes within polycistronic mRNAs and synthetic circuits. Application to lycopene biosynthesis optimization shows robust pathway rewiring and improved yields alongside fine-tuned modulation of essential and competing pathways in Escherichia coli. Our work provides a versatile RNA-regulatory toolkit for next-generation microbial synthetic biology and RNA-based biotechnology.