Targeted DNA integration without double strand breaks using CRISPR RNA guided transposons

Dr. Sternberg shared methods for targeted DNA integration without double-strand breaks, which rely on CRISPR RNA-guided transposons. The use of RNA-guided transposases/recombinases represents an additional tool that may be leveraged to insert large DNA payloads. Transposons are genetic elements containing several genes flanked by conserved inverted repeats. Among different cargo genes, a transposon contains a transposase sequence, which encodes for the enzyme involved in the mobilization of the transposon. By binding to conserved inverted repeats at the end of the transposon, the transposase executes transposon mobilization through a cutting and pasting process from a donor to a target DNA site. Dr. Stemberg highlighted that most transposons exhibit high efficiency for DNA mobilization but lack specificity for DNA targeting. The finding that some transposable elements also encode CRISPR/Cas systems, where the Cas lacks nuclease activity, led them to explore the possible role of CRISPR/Cas on DNA integration. His lab-engineered several plasmids, essentially deconstructing the Vibrio cholera CRISPR-transposon into three individual expression vectors, including mini-transposon, transposase, and CRISPR elements. These constructs, when expressed in E. coli could induce targeted DNA integration guided by RNA. Dr. Sternberg described the elucidated mechanisms involved in the CRISPR RNA-guided integration. This system consists of a complex between Cascade, which binds crRNA and genomic DNA, and other subunits. TniQ, one of the subunits, is critical for subsequent transposase and transposon DNA recruitment. Understanding the structure and basic mechanisms by which the cascade complex operates has enabled Sternberg’s team to develop engineering strategies to improve the efficiency and fidelity of transposition. Lastly, he shared how his lab has implemented these new tools to target large DNA payloads into bacterial genomes.