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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

One gene (TIKI1)-KO rabbit embryos and newborn rabbits. (A) Sequenced mutations in the TIKI1 gene in injected embryos. Deletions are indicated by dashes and insertions are indicated in blue. (B) Generation of TIKI1 KO rabbits by the Cas9/gRNA system. The zygotes (n = 30) microinjection with the 200 ng/μL of Cas9 mRNA and 20 ng/μL of gRNA for TIKI1 were transferred to 3 recipient mothers, 2 of which gave birth to 5 live kits. (C) Detailed mutations of the TIKI1 gene in the 5 KO founders. The number of founder KO rabbits is shown in the left column. In A and C, the WT sequence is shown at the top with the target sites in underline, and the sizes of the deletions (-) or insertions (+) are shown in the right column.
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Fig3: One gene (TIKI1)-KO rabbit embryos and newborn rabbits. (A) Sequenced mutations in the TIKI1 gene in injected embryos. Deletions are indicated by dashes and insertions are indicated in blue. (B) Generation of TIKI1 KO rabbits by the Cas9/gRNA system. The zygotes (n = 30) microinjection with the 200 ng/μL of Cas9 mRNA and 20 ng/μL of gRNA for TIKI1 were transferred to 3 recipient mothers, 2 of which gave birth to 5 live kits. (C) Detailed mutations of the TIKI1 gene in the 5 KO founders. The number of founder KO rabbits is shown in the left column. In A and C, the WT sequence is shown at the top with the target sites in underline, and the sizes of the deletions (-) or insertions (+) are shown in the right column.

Mentions: To further confirm high gene KO efficiency in rabbits by the Cas9/gRNA system, we repeated the experiment with the same approach for another gene, TIKI1 [28]. For in vitro testing, we microinjected 10 pronuclear-stage embryos with Cas9 mRNA and gRNA for TIKI1 at the same concentration as IL2rg. A total of 8 blastocysts were obtained and sequenced. Consistent with the results of IL2rg, the modification efficiency was also 100% (8/8) amongst the embryos, and 4 of the 8 embryos were modified in both alleles (50%) (Table 1). The indels of the TIKI1 in the injected embryos ranged from 2 bp insertions to 18 bp deletions (Figure 3A). Thirty embryos injected with 200 ng/μL of Cas9 mRNA and 20 ng/μL of gRNA for TIKI1 were transferred to 3 pseudo-pregnant recipient rabbits. After about 1 month, 2 of 3 recipient mothers were pregnant to term and gave birth to 5 live kits (Figure 3B). PCR-sequencing of the targeted site in rabbits showed that all 5 newborns were modified at the TIKI1 gene site and that 3 out of 5 newborns were biallelically modified. The indels in the founders ranged from 1 bp insertions to 20 bp deletions (Figure 3C). The #1, #2, and #3 TIKI1 KO rabbits were weak at birth and died within 3 days. The #4 and #5 TIKI1 KO rabbits have been alive for over 5 months with apparent normal phenotype and would require further study.Figure 3


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)

One gene (TIKI1)-KO rabbit embryos and newborn rabbits. (A) Sequenced mutations in the TIKI1 gene in injected embryos. Deletions are indicated by dashes and insertions are indicated in blue. (B) Generation of TIKI1 KO rabbits by the Cas9/gRNA system. The zygotes (n = 30) microinjection with the 200 ng/μL of Cas9 mRNA and 20 ng/μL of gRNA for TIKI1 were transferred to 3 recipient mothers, 2 of which gave birth to 5 live kits. (C) Detailed mutations of the TIKI1 gene in the 5 KO founders. The number of founder KO rabbits is shown in the left column. In A and C, the WT sequence is shown at the top with the target sites in underline, 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

Fig3: One gene (TIKI1)-KO rabbit embryos and newborn rabbits. (A) Sequenced mutations in the TIKI1 gene in injected embryos. Deletions are indicated by dashes and insertions are indicated in blue. (B) Generation of TIKI1 KO rabbits by the Cas9/gRNA system. The zygotes (n = 30) microinjection with the 200 ng/μL of Cas9 mRNA and 20 ng/μL of gRNA for TIKI1 were transferred to 3 recipient mothers, 2 of which gave birth to 5 live kits. (C) Detailed mutations of the TIKI1 gene in the 5 KO founders. The number of founder KO rabbits is shown in the left column. In A and C, the WT sequence is shown at the top with the target sites in underline, and the sizes of the deletions (-) or insertions (+) are shown in the right column.
Mentions: To further confirm high gene KO efficiency in rabbits by the Cas9/gRNA system, we repeated the experiment with the same approach for another gene, TIKI1 [28]. For in vitro testing, we microinjected 10 pronuclear-stage embryos with Cas9 mRNA and gRNA for TIKI1 at the same concentration as IL2rg. A total of 8 blastocysts were obtained and sequenced. Consistent with the results of IL2rg, the modification efficiency was also 100% (8/8) amongst the embryos, and 4 of the 8 embryos were modified in both alleles (50%) (Table 1). The indels of the TIKI1 in the injected embryos ranged from 2 bp insertions to 18 bp deletions (Figure 3A). Thirty embryos injected with 200 ng/μL of Cas9 mRNA and 20 ng/μL of gRNA for TIKI1 were transferred to 3 pseudo-pregnant recipient rabbits. After about 1 month, 2 of 3 recipient mothers were pregnant to term and gave birth to 5 live kits (Figure 3B). PCR-sequencing of the targeted site in rabbits showed that all 5 newborns were modified at the TIKI1 gene site and that 3 out of 5 newborns were biallelically modified. The indels in the founders ranged from 1 bp insertions to 20 bp deletions (Figure 3C). The #1, #2, and #3 TIKI1 KO rabbits were weak at birth and died within 3 days. The #4 and #5 TIKI1 KO rabbits have been alive for over 5 months with apparent normal phenotype and would require further study.Figure 3

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