Abstract
Several strategies have been developed to generate targeted gene disruptions in zebrafish. Here we developed a simple targeted gene inactivation strategy in zebrafish using a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. By injecting two simple in vitrosynthesized components [Cas9 mRNA and single guide (sgRNA)] into one-cell-stage embryos, mutations of the target gene could be efficiently generated. We used a codon-optimized version of Cas9 to improve its translation efficiency in zebrafish. In addition, we designed a cloning-free strategy to facilitate the synthesis of sgRNA. The system allows biallelic inactivation of multiple genes simultaneously by co-injecting a mix of sgRNAs with a single Cas9 construct. This fl exible strategy of gene inactivation provides an efficient way to interrogate gene functions and genetic interactions in zebrafish.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 205-217 |
| Number of pages | 13 |
| Journal | Methods in Molecular Biology |
| Volume | 1332 |
| DOIs | |
| State | Published - 2015 |
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Keywords
- Cas9
- CRISPR
- Genome editing
- Mutagenesis
- Zebrafish
ASJC Scopus subject areas
- Molecular Biology
- Genetics
- Medicine(all)
Cite this
Generation of targeted mutations in zebrafish using the CRISPR/Cas system. / Yin, Linlin; Jao, Li En; Chen, Wenbiao.
In: Methods in Molecular Biology, Vol. 1332, 2015, p. 205-217.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Generation of targeted mutations in zebrafish using the CRISPR/Cas system
AU - Yin, Linlin
AU - Jao, Li En
AU - Chen, Wenbiao
PY - 2015
Y1 - 2015
N2 - Several strategies have been developed to generate targeted gene disruptions in zebrafish. Here we developed a simple targeted gene inactivation strategy in zebrafish using a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. By injecting two simple in vitrosynthesized components [Cas9 mRNA and single guide (sgRNA)] into one-cell-stage embryos, mutations of the target gene could be efficiently generated. We used a codon-optimized version of Cas9 to improve its translation efficiency in zebrafish. In addition, we designed a cloning-free strategy to facilitate the synthesis of sgRNA. The system allows biallelic inactivation of multiple genes simultaneously by co-injecting a mix of sgRNAs with a single Cas9 construct. This fl exible strategy of gene inactivation provides an efficient way to interrogate gene functions and genetic interactions in zebrafish.
AB - Several strategies have been developed to generate targeted gene disruptions in zebrafish. Here we developed a simple targeted gene inactivation strategy in zebrafish using a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. By injecting two simple in vitrosynthesized components [Cas9 mRNA and single guide (sgRNA)] into one-cell-stage embryos, mutations of the target gene could be efficiently generated. We used a codon-optimized version of Cas9 to improve its translation efficiency in zebrafish. In addition, we designed a cloning-free strategy to facilitate the synthesis of sgRNA. The system allows biallelic inactivation of multiple genes simultaneously by co-injecting a mix of sgRNAs with a single Cas9 construct. This fl exible strategy of gene inactivation provides an efficient way to interrogate gene functions and genetic interactions in zebrafish.
KW - Cas9
KW - CRISPR
KW - Genome editing
KW - Mutagenesis
KW - Zebrafish
UR - http://www.scopus.com/inward/record.url?scp=84939507321&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84939507321&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-2917-7_16
DO - 10.1007/978-1-4939-2917-7_16
M3 - Article
C2 - 26285757
AN - SCOPUS:84939507321
VL - 1332
SP - 205
EP - 217
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
SN - 1064-3745
ER -