JUL 22, 2021 2:45 PM EDT

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

C.E. Credits: P.A.C.E. CE Florida CE
Speaker

Abstract

Conventional CRISPR–Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a remarkable inversion of this paradigm, in which bacterial transposons have coopted nuclease-deficient CRISPR–Cas systems to catalyze RNA-guided integration of mobile genetic elements into the genome. Programmable transposition requires CRISPR- and transposon-associated molecular machineries, including a novel co-complex between Cascade and the transposition protein TniQ. Donor DNA integration occurs at a fixed distance downstream of target DNA sequences, accommodates variable length genetic payloads, and functions robustly in diverse bacterial species. Deep sequencing experiments reveal highly specific, genome-wide DNA integration across dozens of unique target sites. The discovery of a fully programmable, RNA-guided transposase lays the foundation for kilobase-scale genome engineering that obviates the requirements for DNA double-strand breaks and homologous recombination.

Learning Objectives:

1. Recall CRISPR-Cas systems are naturally repurposes for non-canonical functions other than cleaving RNA and DNA

2. Indicate CRISPR RNA-guided transposases integrate donor DNA without generating DNA double-strand breaks

3. Explain RNA-guided DNA integration proceeds with high-fidelity and readily accommodates kilobase-size genetic payloads


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