Targeted DNA integration in human cells without double-strand breaks using CRISPR RNA-guided transposases

Traditional genome-editing reagents such as CRISPR-Cas9 achieve targeted DNA modification by introducing double-strand breaks (DSBs), thereby stimulating localized DNA repair by endogenous cellular repair factors. While highly effective at generating heterogenous knockout mutations, this approach suffers from undesirable byproducts and an inability to control product purity. Here we develop a system in human cells for programmable, DSB-free DNA integration using Type I CRISPR-associated transposons (CASTs). To adapt our previously described CAST systems, we optimized DNA targeting by the QCascade complex through a comprehensive assessment of protein design, and we developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase, TnsC, to genomic sites targeted by QCascade. After initial detection of plasmid-based transposition, we screened 15 homologous CAST systems from a wide range of bacterial hosts, identified a CAST homolog from Pseudoalteromonas that exhibited improved activity, and increased integration efficiencies through parameter optimization. We further discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, and we propose that this critical accessory factor functions to drive active disassembly of the post-transposition CAST complex, akin to its demonstrated role in Mu transposition. Our work highlights the ability to functionally reconstitute complex, multi-component machineries in human cells, and establishes a strong foundation to realize the full potential of CRISPR-associated transposons for human genome engineering. ### Competing Interest Statement Columbia University has filed a patent application related to this work. S.E.K., P.L.H.V, A.C., and S.H.S. are inventors on other patents and patent applications related to CRISPR Cas systems and uses thereof. A.C. is a scientific advisor for Vor Biopharma and Cellgorithmics and an equity holder in Cellgorithmics. S.H.S. is a cofounder and scientific advisor to Dahlia Biosciences, a scientific advisor to Prime Medicine and CrisprBits, and an equity holder in Dahlia Biosciences and CrisprBits.