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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

Two gene-KO rabbits obtained by the Cas9/gRNA system in a single step.(A) Generation of 2 gene-KO (IL2rg/RAG1) rabbits via the Cas9/gRNA system. Zygotes (n = 67) were microinjected with 200 ng/μL of Cas9 mRNA, 20 ng/μL of gRNA for IL2rg and 20 ng/μL of gRNA for RAG1 and transferred into 5 recipient mothers, 2 of which gave birth to 5 live kits. (B) Detailed mutations of the IL2rg and RAG1 genes in the 5 KO founders. The number of founder KO rabbits is shown in the left column. For each gene, the WT sequence is shown at the top with the target sites in underline, deletions are indicated by dashes and insertions are indicated in blue, and the sizes of the deletions (-) or insertions (+) are shown in the right column.
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Fig4: Two gene-KO rabbits obtained by the Cas9/gRNA system in a single step.(A) Generation of 2 gene-KO (IL2rg/RAG1) rabbits via the Cas9/gRNA system. Zygotes (n = 67) were microinjected with 200 ng/μL of Cas9 mRNA, 20 ng/μL of gRNA for IL2rg and 20 ng/μL of gRNA for RAG1 and transferred into 5 recipient mothers, 2 of which gave birth to 5 live kits. (B) Detailed mutations of the IL2rg and RAG1 genes in the 5 KO founders. The number of founder KO rabbits is shown in the left column. For each gene, the WT sequence is shown at the top with the target sites in underline, deletions are indicated by dashes and insertions are indicated in blue, and the sizes of the deletions (-) or insertions (+) are shown in the right column.

Mentions: We tested the efficiency of 2 gene modification in 1 step using the Cas9/gRNA system in rabbits. IL2rg and RAG1 were chosen as the genes of interest. Rabbits with a deficiency of IL2rg and RAG1 genes are expected to be an important immunodeficient animal model without mature T cells, B cells and NK cells [29]. A total of 67 zygotes co-injected with a mixture of Cas9 mRNA (200 ng/μL), gRNA for IL2rg (20 ng/μL) and gRNA for RAG1 (20 ng/μL) were transferred into 5 surrogate rabbits. After about 1 month of pregnancy, 2 out of 5 recipient mothers developed to term and gave birth to 5 live kits (3 males, 2 females) (Figure 4A). DNA sequencing of the PCR products spanning the targeted site showed that both IL2rg and RAG1 genes were simultaneously modified in all 5 newborns. The RAG1 gene was biallelically modified in all 5 rabbits and IL2rg gene biallelically modified in both female rabbits. Two of the 5 kits carried the same mutation pattern in 2 alleles (homozygous mutation) at the RAG1 locus. Mutations of the IL2rg gene in the founders ranged from 8 bp insertions to 331 bp deletions (Figure 4B, upper), whilst those of the RAG1 gene ranged from 246 bp insertions to 20 bp deletions (Figure 4B, lower). The #1, and #2 IL2rg/RAG1 KO rabbits were weak at birth and died within 2 days. The other 3 IL2rg/RAG1 KO rabbits died within 2 months (because of infection) when kept in cleaner conditions in isolation after weaning. The thymus phenotype of these KO rabbits was similar to IL2rg KO rabbits (data not shown).Figure 4


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)

Two gene-KO rabbits obtained by the Cas9/gRNA system in a single step.(A) Generation of 2 gene-KO (IL2rg/RAG1) rabbits via the Cas9/gRNA system. Zygotes (n = 67) were microinjected with 200 ng/μL of Cas9 mRNA, 20 ng/μL of gRNA for IL2rg and 20 ng/μL of gRNA for RAG1 and transferred into 5 recipient mothers, 2 of which gave birth to 5 live kits. (B) Detailed mutations of the IL2rg and RAG1 genes in the 5 KO founders. The number of founder KO rabbits is shown in the left column. For each gene, the WT sequence is shown at the top with the target sites in underline, deletions are indicated by dashes and insertions are indicated in blue, and the sizes of the deletions (-) or insertions (+) are shown in the right column.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Two gene-KO rabbits obtained by the Cas9/gRNA system in a single step.(A) Generation of 2 gene-KO (IL2rg/RAG1) rabbits via the Cas9/gRNA system. Zygotes (n = 67) were microinjected with 200 ng/μL of Cas9 mRNA, 20 ng/μL of gRNA for IL2rg and 20 ng/μL of gRNA for RAG1 and transferred into 5 recipient mothers, 2 of which gave birth to 5 live kits. (B) Detailed mutations of the IL2rg and RAG1 genes in the 5 KO founders. The number of founder KO rabbits is shown in the left column. For each gene, the WT sequence is shown at the top with the target sites in underline, deletions are indicated by dashes and insertions are indicated in blue, and the sizes of the deletions (-) or insertions (+) are shown in the right column.
Mentions: We tested the efficiency of 2 gene modification in 1 step using the Cas9/gRNA system in rabbits. IL2rg and RAG1 were chosen as the genes of interest. Rabbits with a deficiency of IL2rg and RAG1 genes are expected to be an important immunodeficient animal model without mature T cells, B cells and NK cells [29]. A total of 67 zygotes co-injected with a mixture of Cas9 mRNA (200 ng/μL), gRNA for IL2rg (20 ng/μL) and gRNA for RAG1 (20 ng/μL) were transferred into 5 surrogate rabbits. After about 1 month of pregnancy, 2 out of 5 recipient mothers developed to term and gave birth to 5 live kits (3 males, 2 females) (Figure 4A). DNA sequencing of the PCR products spanning the targeted site showed that both IL2rg and RAG1 genes were simultaneously modified in all 5 newborns. The RAG1 gene was biallelically modified in all 5 rabbits and IL2rg gene biallelically modified in both female rabbits. Two of the 5 kits carried the same mutation pattern in 2 alleles (homozygous mutation) at the RAG1 locus. Mutations of the IL2rg gene in the founders ranged from 8 bp insertions to 331 bp deletions (Figure 4B, upper), whilst those of the RAG1 gene ranged from 246 bp insertions to 20 bp deletions (Figure 4B, lower). The #1, and #2 IL2rg/RAG1 KO rabbits were weak at birth and died within 2 days. The other 3 IL2rg/RAG1 KO rabbits died within 2 months (because of infection) when kept in cleaner conditions in isolation after weaning. The thymus phenotype of these KO rabbits was similar to IL2rg KO rabbits (data not shown).Figure 4

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