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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 (IL2rg)-KO rabbit embryos and newborn rabbits. (A) Sequenced mutations in the IL2rg gene in injected embryos. Deletions are indicated by dashes, insertions are indicated in blue and substitutions are indicated in pink. (B) Generation of IL2rg KO rabbits via the Cas9/gRNA system. Zygotes (n = 66) microinjected with 200 ng/μL of Cas9 mRNA and 20 ng/μL of gRNA for IL2rg were transferred to 5 recipient mothers, 3 of which gave birth to 8 live kits. (C) Detailed mutations of the IL2rg gene in the 8 KO founders. The number of founder KO rabbits is shown in the left column. (D) Picture of 26 days old IL2rg KO rabbits. (E) The thymus of IL2rg KO rabbits was obviously smaller than that from age-matched WT ones. In A and C, the WT sequence is shown at the top with the target sites in underline; the sizes of the deletions (-) or insertions (+) are shown in the right column. At least 8 TA-clones for each embryos or KO rabbit were used for sequencing to obtain detailed information of the mutation.
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Fig2: One gene (IL2rg)-KO rabbit embryos and newborn rabbits. (A) Sequenced mutations in the IL2rg gene in injected embryos. Deletions are indicated by dashes, insertions are indicated in blue and substitutions are indicated in pink. (B) Generation of IL2rg KO rabbits via the Cas9/gRNA system. Zygotes (n = 66) microinjected with 200 ng/μL of Cas9 mRNA and 20 ng/μL of gRNA for IL2rg were transferred to 5 recipient mothers, 3 of which gave birth to 8 live kits. (C) Detailed mutations of the IL2rg gene in the 8 KO founders. The number of founder KO rabbits is shown in the left column. (D) Picture of 26 days old IL2rg KO rabbits. (E) The thymus of IL2rg KO rabbits was obviously smaller than that from age-matched WT ones. In A and C, the WT sequence is shown at the top with the target sites in underline; the sizes of the deletions (-) or insertions (+) are shown in the right column. At least 8 TA-clones for each embryos or KO rabbit were used for sequencing to obtain detailed information of the mutation.

Mentions: We selected the IL2rg gene (X-linked) as the first gene of interest to test the efficiency of 1 gene-KO rabbits. A mixture of Cas9 mRNA and gRNA for IL2rg was microinjected into the cytoplasm of pronuclear-stage embryos with working concentrations of 200 ng/μL of Cas9 mRNA and 20 ng/μL of gRNA. Sixteen of 21 injected embryos developed into the blastocyst stage, and PCR products derived from 12 blastocysts were sequenced to confirm mutation efficiency. As shown in Table 1, IL2rg mutation was found in all 12 embryos. More strikingly, we detected no wild type (WT) sequence among the 12 embryos. The indels of IL2rg gene ranged from 30 base pair (bp) insertions to 21 bp deletions (Figure 2A). We then used this system to produce IL2rg KO rabbits. A total of 66 embryos injected with the same concentration of Cas9 mRNA and gRNA for IL2rg were transferred to 5 pseudo-pregnant recipient rabbits. After about 1 month, 3 of 5 recipient mothers were pregnant to term and gave birth to 8 live kits (5 males, 3 females) (Figure 2B). PCR-sequencing of the targeted site in rabbits showed that all 5 male newborns were IL2rg KO in X chromosome and all 3 female newborns were mutated in both X chromosomes. The indels in the founders ranged from 9 bp to 321 bp deletions (Figure 2C). All IL2rg KO rabbits were alive for no more than 45 days because of diarrhea, pulmonary infection or other causes, except the #2 (female, lived for 229 days) rabbit, kept in conventional housing conditions (Figure 2D). We performed the autopsies soon after their death and found that IL2rg KO rabbits had undersized thymuses compared with age-matched WT ones (Figure 2E).Table 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)

One gene (IL2rg)-KO rabbit embryos and newborn rabbits. (A) Sequenced mutations in the IL2rg gene in injected embryos. Deletions are indicated by dashes, insertions are indicated in blue and substitutions are indicated in pink. (B) Generation of IL2rg KO rabbits via the Cas9/gRNA system. Zygotes (n = 66) microinjected with 200 ng/μL of Cas9 mRNA and 20 ng/μL of gRNA for IL2rg were transferred to 5 recipient mothers, 3 of which gave birth to 8 live kits. (C) Detailed mutations of the IL2rg gene in the 8 KO founders. The number of founder KO rabbits is shown in the left column. (D) Picture of 26 days old IL2rg KO rabbits. (E) The thymus of IL2rg KO rabbits was obviously smaller than that from age-matched WT ones. In A and C, the WT sequence is shown at the top with the target sites in underline; the sizes of the deletions (-) or insertions (+) are shown in the right column. At least 8 TA-clones for each embryos or KO rabbit were used for sequencing to obtain detailed information of the mutation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: One gene (IL2rg)-KO rabbit embryos and newborn rabbits. (A) Sequenced mutations in the IL2rg gene in injected embryos. Deletions are indicated by dashes, insertions are indicated in blue and substitutions are indicated in pink. (B) Generation of IL2rg KO rabbits via the Cas9/gRNA system. Zygotes (n = 66) microinjected with 200 ng/μL of Cas9 mRNA and 20 ng/μL of gRNA for IL2rg were transferred to 5 recipient mothers, 3 of which gave birth to 8 live kits. (C) Detailed mutations of the IL2rg gene in the 8 KO founders. The number of founder KO rabbits is shown in the left column. (D) Picture of 26 days old IL2rg KO rabbits. (E) The thymus of IL2rg KO rabbits was obviously smaller than that from age-matched WT ones. In A and C, the WT sequence is shown at the top with the target sites in underline; the sizes of the deletions (-) or insertions (+) are shown in the right column. At least 8 TA-clones for each embryos or KO rabbit were used for sequencing to obtain detailed information of the mutation.
Mentions: We selected the IL2rg gene (X-linked) as the first gene of interest to test the efficiency of 1 gene-KO rabbits. A mixture of Cas9 mRNA and gRNA for IL2rg was microinjected into the cytoplasm of pronuclear-stage embryos with working concentrations of 200 ng/μL of Cas9 mRNA and 20 ng/μL of gRNA. Sixteen of 21 injected embryos developed into the blastocyst stage, and PCR products derived from 12 blastocysts were sequenced to confirm mutation efficiency. As shown in Table 1, IL2rg mutation was found in all 12 embryos. More strikingly, we detected no wild type (WT) sequence among the 12 embryos. The indels of IL2rg gene ranged from 30 base pair (bp) insertions to 21 bp deletions (Figure 2A). We then used this system to produce IL2rg KO rabbits. A total of 66 embryos injected with the same concentration of Cas9 mRNA and gRNA for IL2rg were transferred to 5 pseudo-pregnant recipient rabbits. After about 1 month, 3 of 5 recipient mothers were pregnant to term and gave birth to 8 live kits (5 males, 3 females) (Figure 2B). PCR-sequencing of the targeted site in rabbits showed that all 5 male newborns were IL2rg KO in X chromosome and all 3 female newborns were mutated in both X chromosomes. The indels in the founders ranged from 9 bp to 321 bp deletions (Figure 2C). All IL2rg KO rabbits were alive for no more than 45 days because of diarrhea, pulmonary infection or other causes, except the #2 (female, lived for 229 days) rabbit, kept in conventional housing conditions (Figure 2D). We performed the autopsies soon after their death and found that IL2rg KO rabbits had undersized thymuses compared with age-matched WT ones (Figure 2E).Table 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