Multiplex Genome Engineering Using CRISPR/Cas Systems

TLDR

Precise genome editing is essential for functional elucidation of causal genetic variants, and the type II CRISPR/Cas system enables RNA‑guided site‑specific DNA cleavage. The study engineered two type II CRISPR/Cas systems to demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. They engineered two type II CRISPR/Cas systems, using short RNAs to guide Cas9 nucleases to cleave endogenous genomic loci in human and mouse cells. Cas9 can be converted into a nicking enzyme to promote homology‑directed repair with minimal mutagenic activity, and multiple guide sequences can be encoded in a single CRISPR array to enable simultaneous editing of several mammalian genomic sites, showing the technology’s programmability and broad applicability.

Abstract

Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.