Programmable, multiplexed and orthogonal gene control in bacteria with attenuated Cas13d systems

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.