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Generation of multi-gene knockout rabbits using the Cas9/gRNA system.

Yan Q, Zhang Q, Yang H, Zou Q, Tang C, Fan N, Lai L - Cell Regen (Lond) (2014)

Bottom Line: In this report, we applied CRISPR technology to rabbits by microinjection of Cas9 mRNA and guided RNA (gRNA) into the cytoplasm of pronuclear-stage embryos.We also tested the efficiency of multiple gene KOs in early rabbit embryos and found that the efficiency of simultaneous gene mutation on target sites is as high as 100% for 3 genes (IL2rg, RAG1 and RAG2) and 33.3% for 5 genes (IL2rg, RAG1, RAG2, TIKI1 and ALB).Our results demonstrate that the Cas9/gRNA system is a highly efficient and fast tool not only for single-gene editing but also for multi-gene editing in rabbits.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

ABSTRACT
The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) is a simple, robust and efficient technique for gene targeting in model organisms such as zebrafish, mice and rats. In this report, we applied CRISPR technology to rabbits by microinjection of Cas9 mRNA and guided RNA (gRNA) into the cytoplasm of pronuclear-stage embryos. We achieved biallelic gene knockout (KO) rabbits by injection of 1 gene (IL2rg) or 2 gene (IL2rg and RAG1) Cas9 mRNA and gRNA with an efficiency of 100%. We also tested the efficiency of multiple gene KOs in early rabbit embryos and found that the efficiency of simultaneous gene mutation on target sites is as high as 100% for 3 genes (IL2rg, RAG1 and RAG2) and 33.3% for 5 genes (IL2rg, RAG1, RAG2, TIKI1 and ALB). Our results demonstrate that the Cas9/gRNA system is a highly efficient and fast tool not only for single-gene editing but also for multi-gene editing in rabbits.

No MeSH data available.


Related in: MedlinePlus

Genome editing via the Cas9/gRNA system in rabbit. (A) Constructs and schematic illustration of the Cas9/gRNA system used in this study. The T7 promoter drives transcription of the gRNA, consisting of a target sequence and a scaffold sequence. NLS: nuclear localization signal. bGH polyA: bovine growth hormone poly-A. (B) Target sequence of the IL2rg, RAG1, RAG2, TIKI1 and ALB genes using the Cas9/gRNA system. gRNA-targeting sequences are underlined and PAM sequences are highlighted in red.
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Fig1: Genome editing via the Cas9/gRNA system in rabbit. (A) Constructs and schematic illustration of the Cas9/gRNA system used in this study. The T7 promoter drives transcription of the gRNA, consisting of a target sequence and a scaffold sequence. NLS: nuclear localization signal. bGH polyA: bovine growth hormone poly-A. (B) Target sequence of the IL2rg, RAG1, RAG2, TIKI1 and ALB genes using the Cas9/gRNA system. gRNA-targeting sequences are underlined and PAM sequences are highlighted in red.

Mentions: We constructed a vector with the T7 promoter that can be in vitro transcribed into Cas9 mRNA and gRNA (Figure 1A). The sequences of all target sites (22 nucleotides or 23 nucleotides) (Figure 1B) begin with GG at the 5’ end because transcription is initiated from GG bases and ended with the protospacer adjacent motif (PAM) NGG at the 3’ end, which is indispensable for Cas9 binding and cleavage [27].Figure 1


Generation of multi-gene knockout rabbits using the Cas9/gRNA system.

Yan Q, Zhang Q, Yang H, Zou Q, Tang C, Fan N, Lai L - Cell Regen (Lond) (2014)

Genome editing via the Cas9/gRNA system in rabbit. (A) Constructs and schematic illustration of the Cas9/gRNA system used in this study. The T7 promoter drives transcription of the gRNA, consisting of a target sequence and a scaffold sequence. NLS: nuclear localization signal. bGH polyA: bovine growth hormone poly-A. (B) Target sequence of the IL2rg, RAG1, RAG2, TIKI1 and ALB genes using the Cas9/gRNA system. gRNA-targeting sequences are underlined and PAM sequences are highlighted in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230364&req=5

Fig1: Genome editing via the Cas9/gRNA system in rabbit. (A) Constructs and schematic illustration of the Cas9/gRNA system used in this study. The T7 promoter drives transcription of the gRNA, consisting of a target sequence and a scaffold sequence. NLS: nuclear localization signal. bGH polyA: bovine growth hormone poly-A. (B) Target sequence of the IL2rg, RAG1, RAG2, TIKI1 and ALB genes using the Cas9/gRNA system. gRNA-targeting sequences are underlined and PAM sequences are highlighted in red.
Mentions: We constructed a vector with the T7 promoter that can be in vitro transcribed into Cas9 mRNA and gRNA (Figure 1A). The sequences of all target sites (22 nucleotides or 23 nucleotides) (Figure 1B) begin with GG at the 5’ end because transcription is initiated from GG bases and ended with the protospacer adjacent motif (PAM) NGG at the 3’ end, which is indispensable for Cas9 binding and cleavage [27].Figure 1

Bottom Line: In this report, we applied CRISPR technology to rabbits by microinjection of Cas9 mRNA and guided RNA (gRNA) into the cytoplasm of pronuclear-stage embryos.We also tested the efficiency of multiple gene KOs in early rabbit embryos and found that the efficiency of simultaneous gene mutation on target sites is as high as 100% for 3 genes (IL2rg, RAG1 and RAG2) and 33.3% for 5 genes (IL2rg, RAG1, RAG2, TIKI1 and ALB).Our results demonstrate that the Cas9/gRNA system is a highly efficient and fast tool not only for single-gene editing but also for multi-gene editing in rabbits.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

ABSTRACT
The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) is a simple, robust and efficient technique for gene targeting in model organisms such as zebrafish, mice and rats. In this report, we applied CRISPR technology to rabbits by microinjection of Cas9 mRNA and guided RNA (gRNA) into the cytoplasm of pronuclear-stage embryos. We achieved biallelic gene knockout (KO) rabbits by injection of 1 gene (IL2rg) or 2 gene (IL2rg and RAG1) Cas9 mRNA and gRNA with an efficiency of 100%. We also tested the efficiency of multiple gene KOs in early rabbit embryos and found that the efficiency of simultaneous gene mutation on target sites is as high as 100% for 3 genes (IL2rg, RAG1 and RAG2) and 33.3% for 5 genes (IL2rg, RAG1, RAG2, TIKI1 and ALB). Our results demonstrate that the Cas9/gRNA system is a highly efficient and fast tool not only for single-gene editing but also for multi-gene editing in rabbits.

No MeSH data available.


Related in: MedlinePlus