RNA-Guided Human Genome Engineering via Cas9

By P. Mali, Luhan Yang, Kevin M. Esvelt, John D Aach, M. Guell, James E. DiCarlo, J. Norville, George M. Church. 2013

9,023 citations recorded by the source corpus.

Genome Editing Clustered regularly interspaced short palindromic repeats (CRISPR) function as part of an adaptive immune system in a range of prokaryotes: Invading phage and plasmid DNA is targeted for cleavage by complementary CRISPR RNAs (crRNAs) bound to a CRISPR-associated endonuclease (see the Perspective by van der Oost). Cong et al. (p. 819, published online 3 January) and Mali et al. (p. 823, published online 3 January) adapted this defense system to function as a genome editing tool in eukaryotic cells. A bacterial genome defense system is adapted to function as a genome-editing tool in mammalian cells. [Also see Perspective by van der Oost] 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.

Subfields: Genetics, Molecular Biology

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Citations in the corpus

mRNA-based therapeutics — developing a new class of drugs (2014, neutral): “Custom-designed zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR–Cas9 (clustered regularly spaced short palindromic repeat– CRISPR-associated protein 9) provide powerful tools for site-specific modification of genomes 164–166 .”
Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition (2016, background): “org Clin Cancer Res; 23(9) May 1, 2017 2257”
Cas-analyzer: an online tool for assessing genome editing results using NGS data (2016, background): “Currently, Cas-Analyzer supports various programmable nucleases, including single nucleases and paired nucleases.”
A CRISPR/Cas9 toolkit for multiplex genome editing in plants (2014, methodology): “The CRISPR/Cas system has been harnessed to achieve efficient genome editing in a variety of organisms, including bacteria, yeast, plants, and animals, as well as human cell lines [12-27].”
The CRISPR/Cas9 system for plant genome editing and beyond. (2015, background): “Importantly, it was also shown that multiple gRNAs with different sequences could be used to achieve high-efficiency multiplex genome engineering at different loci simultaneously (Cong et al., 2013; Mali et al., 2013).”
Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide RNAs (2014, methodology): “Cas9 cloning and protein expression For in vivo expression of Cas9, the Cas9 open reading frame was amplified from hCas9 [5] and cloned into the pCS2 vector to generate pCS2-Cas9.”
CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture. (2019, background): “Type II CRISPR/Cas9 from Streptococcus pyogenes was the first system shown to specifically cleave DNA in vitro and in vivo (21, 36, 54, 103).”
CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes (2015, background): “Unintended mutation in the genome can greatly hinder the application of CRISPR/Cas9, especially in studies of development and gene therapy (Hsu et al., 2014; Mali et al., 2013b; Sander and Joung, 2014).”
Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018, background): “Since first applied in mammalian cells in 2013, CRISPR/Cas system, based on a RNA-guided nuclease, has revolutionized the way that genome editing is performed [5, 6].”
CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences (2014, positive): “Chimeric single-guided RNAs (sgRNAs) based on CRISPR (9) have been engineered to direct the Cas9 nuclease to cleave complementary genomic sequences when followed by a 5 -NGG protospacer-adjacent motif (PAM) in eukaryotic cells (10–12).”
CRISPR/Cas Systems towards Next-Generation Biosensing. (2019, neutral): “Timeline : First CRISPR/Cas9 gene edi ng system [5,8]”