RNA-Guided Human Genome Engineering via Cas9

TLDR

CRISPR/Cas systems in bacteria and archaea use short RNA guides to target and degrade foreign nucleic acids. The authors engineered a type II CRISPR system to function with custom guide RNAs in human cells. They adapted the bacterial system for human cells and generated a genome‑wide library of ~190 000 unique gRNAs covering ~40 % of human exons. The engineered system achieved 2–25 % targeting at the AAVS1 locus across cell lines, proved sequence‑specific, enabled multiplex editing, and demonstrates a robust, multiplexable RNA‑guided tool for human genome engineering.

Abstract

Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.