Summary CRISPR-Cas systems are a powerful gene editing technique. However, their wide applications are limited by off-target effects and low efficiency of delivering large genome-editing tools. Here we report a novel family of RNA-guided DNA nucleases. Bioinformatics analysis reveals stable co-occurrence of conserved RAGATH-18-associated RNAs (raRNAs) and their upstream IS607 OrfBs with an average of 390 amino acids. raRNAs interact with OrfBs to form programmable raRNA-Associated DNases (RADs). The RAD systems display robust dsDNA interference activity in bacteria and human cells. dsDNA cleavage activity of the new systems is highly sensitive to mismatches between the guide and target sequences. A cryo-EM structure of the RAD effector protein from Firmicutes bacteria (FbRAD1) bound by its cognate raRNA and a dsDNA target reveals the mechanisms of raRNA-guided RNA targeting of the RAD systems. Together, the study identifies a novel family of compact RNA-guided DNases with potentials of application in gene editing.

Download information

The following table provides the small RNA sequencing.

Description	Download
Raw Read1 of FbRAD1
Raw Read2 of FbRAD1
Md5 file for Raw Read1 of FbRAD1
Md5 file for Raw Read2 of FbRAD1