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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

Off-target analysis of Cas9/gRNA system-induced mutations in KO founders. (A) T7EI assays of the PCR products of candidate off-target sites using the pooled DNA of all KO founders for each gene as the template (primer sequences listed in Additional file 3: Table S2). The IOT5 could be cleaved (red arrowheads). (B) Sequencing diagram of IOT5 in #1 IL2rg KO new-born rabbits showing a double curve after the mutation around the PAM region. (C) Detailed mutations of IOT5 in the newborn rabbits. The number of founder KO rabbits is shown in the left column. The WT sequence is shown at the top with the target sites in underline. Deletions are indicated by dashes, insertions are indicated in blue and substitutions are indicated in pink, and the sizes of the deletions (-) or insertions (+) are shown in the right column. The fractions indicate the read number of the mutant allele (numerator) out of total read number (denominator).
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Fig5: Off-target analysis of Cas9/gRNA system-induced mutations in KO founders. (A) T7EI assays of the PCR products of candidate off-target sites using the pooled DNA of all KO founders for each gene as the template (primer sequences listed in Additional file 3: Table S2). The IOT5 could be cleaved (red arrowheads). (B) Sequencing diagram of IOT5 in #1 IL2rg KO new-born rabbits showing a double curve after the mutation around the PAM region. (C) Detailed mutations of IOT5 in the newborn rabbits. The number of founder KO rabbits is shown in the left column. The WT sequence is shown at the top with the target sites in underline. Deletions are indicated by dashes, insertions are indicated in blue and substitutions are indicated in pink, and the sizes of the deletions (-) or insertions (+) are shown in the right column. The fractions indicate the read number of the mutant allele (numerator) out of total read number (denominator).

Mentions: Several studies have suggested that the 8–12 bases (seed sequence) of the gRNA sequence close to PAM and PAM itself are critical factors determining site-specific cleavage. In other words, slight mismatches beyond the seed sequence may lead to off-target cleavage [27]. To detect the occurrence of off-target mutations in all 18 newborn rabbits (8 IL2rg KO rabbits, 5 TIKI1 KO rabbits and 5 IL2rg/RAG1 KO rabbits), we selected candidate sites with over 14 bases identical to the targeted sites of IL2rg, TIKI1 and RAG1. Nine sites (IOT1 to IOT9) were determined to be potential off-targeting sites for the IL2rg gene, 17 sites (TOT1 to TOT17) were determined to be potential off-targeting sites for the TIKI1 gene, whilst 3 sites (ROT1 to ROT3) were determined to be potential off-targeting sites for the RAG1 gene in the rabbit genome (Additional file 2: Figure S2). T7 endonuclease I (T7EI) assay showed that only the IOT5 site presented detectable mutagenic activity amongst these 29 potential off-target sites in all founder rabbits (Figure 5A). To confirm the results of T7EI assay, we further examined the DNA sequences of PCR products. Mutations at the IOT5 site occurred around the PAM region (Figure 5B). DNA sequencing showed that 5 (#1, #3, #5, #6, #2-4) of 13 founders had mutations at the IOT5 site. Details of these mutations are shown in Figure 5C.Figure 5


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)

Off-target analysis of Cas9/gRNA system-induced mutations in KO founders. (A) T7EI assays of the PCR products of candidate off-target sites using the pooled DNA of all KO founders for each gene as the template (primer sequences listed in Additional file 3: Table S2). The IOT5 could be cleaved (red arrowheads). (B) Sequencing diagram of IOT5 in #1 IL2rg KO new-born rabbits showing a double curve after the mutation around the PAM region. (C) Detailed mutations of IOT5 in the newborn rabbits. The number of founder KO rabbits is shown in the left column. The WT sequence is shown at the top with the target sites in underline. Deletions are indicated by dashes, insertions are indicated in blue and substitutions are indicated in pink, and the sizes of the deletions (-) or insertions (+) are shown in the right column. The fractions indicate the read number of the mutant allele (numerator) out of total read number (denominator).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: Off-target analysis of Cas9/gRNA system-induced mutations in KO founders. (A) T7EI assays of the PCR products of candidate off-target sites using the pooled DNA of all KO founders for each gene as the template (primer sequences listed in Additional file 3: Table S2). The IOT5 could be cleaved (red arrowheads). (B) Sequencing diagram of IOT5 in #1 IL2rg KO new-born rabbits showing a double curve after the mutation around the PAM region. (C) Detailed mutations of IOT5 in the newborn rabbits. The number of founder KO rabbits is shown in the left column. The WT sequence is shown at the top with the target sites in underline. Deletions are indicated by dashes, insertions are indicated in blue and substitutions are indicated in pink, and the sizes of the deletions (-) or insertions (+) are shown in the right column. The fractions indicate the read number of the mutant allele (numerator) out of total read number (denominator).
Mentions: Several studies have suggested that the 8–12 bases (seed sequence) of the gRNA sequence close to PAM and PAM itself are critical factors determining site-specific cleavage. In other words, slight mismatches beyond the seed sequence may lead to off-target cleavage [27]. To detect the occurrence of off-target mutations in all 18 newborn rabbits (8 IL2rg KO rabbits, 5 TIKI1 KO rabbits and 5 IL2rg/RAG1 KO rabbits), we selected candidate sites with over 14 bases identical to the targeted sites of IL2rg, TIKI1 and RAG1. Nine sites (IOT1 to IOT9) were determined to be potential off-targeting sites for the IL2rg gene, 17 sites (TOT1 to TOT17) were determined to be potential off-targeting sites for the TIKI1 gene, whilst 3 sites (ROT1 to ROT3) were determined to be potential off-targeting sites for the RAG1 gene in the rabbit genome (Additional file 2: Figure S2). T7 endonuclease I (T7EI) assay showed that only the IOT5 site presented detectable mutagenic activity amongst these 29 potential off-target sites in all founder rabbits (Figure 5A). To confirm the results of T7EI assay, we further examined the DNA sequences of PCR products. Mutations at the IOT5 site occurred around the PAM region (Figure 5B). DNA sequencing showed that 5 (#1, #3, #5, #6, #2-4) of 13 founders had mutations at the IOT5 site. Details of these mutations are shown in Figure 5C.Figure 5

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