RNA-Guided Human Genome Engineering via Cas9¶
Why this mattered¶
This paper mattered because it helped turn CRISPR-Cas9 from a bacterial immune mechanism into a practical, programmable tool for editing human genomes. Earlier genome-engineering platforms such as zinc-finger nucleases and TALENs required custom protein engineering for each target site. Mali et al. showed that, in human cells, target choice could instead be redirected by changing a short guide RNA. That shifted the limiting step from protein design to RNA design, making genome editing simpler, cheaper, faster, and more scalable.
The paper also demonstrated features that became central to the CRISPR era: efficient editing at an endogenous human locus, activity in several human cell types including induced pluripotent stem cells, sequence-specific targeting, and multiplex editing by introducing multiple guide RNAs at once. Its genome-wide guide RNA resource underscored a new way of thinking about genome engineering: not as a bespoke intervention at one locus, but as a broadly programmable platform for perturbing many genes across the human genome.
In retrospect, the importance of the work lies less in any single target edited than in the experimental regime it made credible. After this paper and closely related 2013 demonstrations, CRISPR-Cas9 rapidly became the default tool for functional genomics, pooled genetic screens, disease modeling, cellular engineering, and eventually therapeutic genome editing. Later breakthroughs such as CRISPR screening libraries, base editing, prime editing, epigenome editing, and ex vivo CRISPR therapies all built on the same paradigm established here: a nuclease or editor can be brought to a chosen genomic address by an easily reprogrammable RNA.
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.
Related¶
- cite → A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity — RNA-guided human genome engineering applies the dual-RNA Cas9 endonuclease mechanism characterized by Jinek et al. to editing human cells.
- cite → Multiplex Genome Engineering Using CRISPR/Cas Systems — RNA-guided human genome engineering is linked to multiplex CRISPR/Cas genome engineering through programmable Cas9 targeting of multiple genomic loci.
- cite → Ultrafast and memory-efficient alignment of short DNA sequences to the human genome — Cas9 genome editing used Bowtie-style short-read alignment to map sequencing reads that verified targeted human genome modifications.
- cite ← Genome engineering using the CRISPR-Cas9 system — Both papers demonstrate Cas9 as an RNA-guided tool for targeted genome editing in human cells.
- cite ← Multiplex Genome Engineering Using CRISPR/Cas Systems — The multiplex CRISPR paper cites RNA-guided human genome engineering via Cas9 as evidence that Cas9 can be programmed for targeted editing in mammalian cells.