Work

Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9

Year: 2015

Type: review

Abstract: Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid offtarget mutations.1 PROGRAMMABLE NUCLEASES FOR GENOME EDITINGGenome editing is a method of modifying genome sequences in cells and whole organisms via custom-designed programmable nucleases (Kim and Kim, 2014), which cleave chromosomal DNA in a targeted manner, producing site-specific DNA double-strand breaks (DSBs).These DSBs are efficiently repaired in cells by endogenous DNA repair systems known as homologous recombination (HR) and non-homologous end joining (NHEJ), often causing site-specific genome modifications.This technique is now widely used in research, medicine, and biotechnology, a phenomenon that is highlighted by the choice of genome editing as the 2011 Method of the Year and one of the 2015 Methods to Watch by Nature Methods and as a Breakthrough of the Year runner-up by Science in 2013.For example, gene knockout using engineered nucleases enables identification and validation of drug-able target genes.Gene correction in stem and somatic cells can lead to gene therapy

Source: Molecules and Cells

DOI: 10.14348/molcells.2015.0103

Authors: Taeyoung Koo, Jungjoon Lee, Jin-Soo Kim

Institutions: Institute for Basic Science, Korea University of Science and Technology, Seoul National University, Institute of Molecular Biology and Genetics

Language: English

Topic: CRISPR and Genetic Engineering

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $2,500