TY - JOUR

T1 - Guide RNA modification as a way to improve CRISPR/Cas9-based genome-editing systems

AU - Filippova, Julia

AU - Matveeva, Anastasiya

AU - Zhuravlev, Evgenii

AU - Stepanov, Grigory

N1 - Publisher Copyright: © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM) Copyright: Copyright 2019 Elsevier B.V., All rights reserved.

PY - 2019/12/1

Y1 - 2019/12/1

N2 - Genome-editing technologies, in particular, CRISPR systems, are widely used for targeted regulation of gene expression and obtaining modified human and animal cell lines, plants, fungi, and animals with preassigned features. Despite being well described and easy to perform, the most common methods for construction and delivery of CRISPR/Cas9-containing plasmid systems possess significant disadvantages, mostly associated with effects of the presence of exogenous DNA within the cell. Transfection with active ribonucleoprotein complexes of Cas9 with single-guide RNAs (sgRNAs) represents one of the most promising options because of faster production of sgRNAs, the ability of a researcher to control the amount of sgRNA delivered into the cell, and consequently, fewer off-target mutations. Artificial-RNA synthesis strategies allow for the introduction of various modified components, such as backbone alterations, native structural motifs, and labels for visualization. Modifications of RNA can increase its resistance to hydrolysis, alter the thermodynamic stability of RNA–protein and RNA–DNA complexes, and reduce the immunogenic and cytotoxic effects. This review describes various approaches to improving synthetic guide RNA function through nucleotide modification.

AB - Genome-editing technologies, in particular, CRISPR systems, are widely used for targeted regulation of gene expression and obtaining modified human and animal cell lines, plants, fungi, and animals with preassigned features. Despite being well described and easy to perform, the most common methods for construction and delivery of CRISPR/Cas9-containing plasmid systems possess significant disadvantages, mostly associated with effects of the presence of exogenous DNA within the cell. Transfection with active ribonucleoprotein complexes of Cas9 with single-guide RNAs (sgRNAs) represents one of the most promising options because of faster production of sgRNAs, the ability of a researcher to control the amount of sgRNA delivered into the cell, and consequently, fewer off-target mutations. Artificial-RNA synthesis strategies allow for the introduction of various modified components, such as backbone alterations, native structural motifs, and labels for visualization. Modifications of RNA can increase its resistance to hydrolysis, alter the thermodynamic stability of RNA–protein and RNA–DNA complexes, and reduce the immunogenic and cytotoxic effects. This review describes various approaches to improving synthetic guide RNA function through nucleotide modification.

KW - CRISPR/Cas9

KW - Genome editing

KW - Guide RNA

KW - RNA modification

KW - Single-guide RNA

KW - MUTAGENESIS

KW - COMPLEX

KW - SPECIFICITY

KW - HUMAN-CELLS

KW - TARGET DNA

KW - CLEAVAGE

KW - GENE

KW - PSEUDOURIDINE

KW - CAS9

KW - REPEATS

UR - http://www.scopus.com/inward/record.url?scp=85072162193&partnerID=8YFLogxK

U2 - 10.1016/j.biochi.2019.09.003

DO - 10.1016/j.biochi.2019.09.003

M3 - Review article

C2 - 31493470

AN - SCOPUS:85072162193

VL - 167

SP - 49

EP - 60

JO - Biochimie

JF - Biochimie

SN - 0300-9084

ER -