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CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA
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CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA
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Journal Article
CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA
2015
Overview
Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous
eukaryotic translation elongation factor 2
(
eEF-2
) gene] using the
C
lustered
R
egularly
I
nterspaced
S
hort
P
alindromic
R
epeats/
C
rispr
as
sociated 9 (CRISPR/Cas9) system. We used
in vitro
synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [
orthodenticle homeobox 2
(
Otx2
)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method.
Publisher
Nature Publishing Group UK,Nature Publishing Group
Subject
/ 42
/ 42/41
/ 64/60
/ Animals
/ Bacterial Proteins - genetics
/ Clustered Regularly Interspaced Short Palindromic Repeats
/ CRISPR
/ DNA, Single-Stranded - genetics
/ Female
/ Gene Knockdown Techniques - methods
/ Genomes
/ Green Fluorescent Proteins - biosynthesis
/ Green Fluorescent Proteins - genetics
/ Homeobox
/ Humanities and Social Sciences
/ Introns
/ Male
/ miRNA
/ Otx Transcription Factors - biosynthesis
/ Otx Transcription Factors - genetics
/ Science
