PUBLICATION
A G-quadruplex motif at the 3' end of sgRNAs improves CRISPR-Cas9 based genome editing efficiency
- Authors
- Nahar, S., Sehgal, P., Azhar, M., Rai, M., Singh, A., Sivasubbu, S., Chakraborty, D., Maiti, S.
- ID
- ZDB-PUB-180223-26
- 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, Kinetoplastida/chemistry
- RNA, Guide, Kinetoplastida/genetics*
- Zebrafish
- PubMed
- 29450416 Full text @ Chem. Commun. (Camb.)
Citation
Nahar, S., Sehgal, P., Azhar, M., Rai, M., Singh, A., Sivasubbu, S., Chakraborty, D., Maiti, S. (2018) A G-quadruplex motif at the 3' end of sgRNAs improves CRISPR-Cas9 based genome editing efficiency. Chemical communications (Cambridge, England). 54(19):2377-2380.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping