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ZIRC
ZFIN ID: ZDB-PUB-180223-26
A G-quadruplex motif at the 3' end of sgRNAs improves CRISPR-Cas9 based genome editing efficiency
Nahar, S., Sehgal, P., Azhar, M., Rai, M., Singh, A., Sivasubbu, S., Chakraborty, D., Maiti, S.
Date: 2018
Source: Chemical communications (Cambridge, England)   54(19): 2377-2380 (Journal)
Registered Authors: Sivasubbu, Sridhar
Keywords: none
MeSH Terms:
  • Animals
  • Bacterial Proteins/chemistry*
  • Bacterial Proteins/metabolism
  • CRISPR-Cas Systems/genetics*
  • Cell Line
  • DNA/chemistry
  • Embryo, Nonmammalian/metabolism
  • Endonucleases/chemistry*
  • Endonucleases/metabolism
  • G-Quadruplexes*
  • Gene Editing*
  • INDEL Mutation
  • Inverted Repeat Sequences
  • Mice
  • RNA/chemistry
  • RNA/genetics*
  • RNA Stability
  • RNA, Guide/chemistry
  • RNA, Guide/genetics*
  • Zebrafish
PubMed: 29450416 Full text @ Chem. Commun. (Camb.)
ABSTRACT
Originating as a component of prokaryotic adaptive immunity, the type II CRISPR/Cas9 system has been repurposed for targeted genome editing in various organisms. Although Cas9 can bind and cleave DNA efficiently under in vitro conditions, its activity inside a cell can vary dramatically between targets owing to the differences between genomic loci and the availability of enough Cas9/sgRNA (single guide RNA) complex molecules for cleavage. Most methods have so far relied on Cas9 protein engineering or base modifications in the sgRNA sequence to improve CRISPR/Cas9 activity. Here we demonstrate that a structure based rational design of sgRNAs can enhance the efficiency of Cas9 cleavage in vivo. By appending a naturally forming RNA G-quadruplex motif to the 3' end of sgRNAs we can improve its stability and target cleavage efficiency in zebrafish embryos without inducing off-target activity, thereby underscoring its value in the design of better and optimized genome editing triggers.
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