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Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs.

Wang X, Zhou J, Cao C, Huang J, Hai T, Wang Y, Zheng Q, Zhang H, Qin G, Miao X, Wang H, Cao S, Zhou Q, Zhao J - Sci Rep (2015)

Bottom Line: Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks.The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%.Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, the uncertainties caused by wide variations in sgRNA activity impede the utility of this system in generating genetically modified pigs. Here, we described a single blastocyst genotyping system to provide a simple and rapid solution to evaluate and compare the sgRNA efficiency at inducing indel mutations for a given gene locus. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%. Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer. We further showed that direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Thus, our method considerably reduces the uncertainties and expands the practical possibilities of CRISPR/Cas9-mediated genome engineering in pigs.

No MeSH data available.


Design and construction of CRISPR.(A) Schematic of the Cas9/sgRNA-targeting sites in pig mitfloci. Exons are shown as black boxes. The sgRNA-targeting sequence is labeled in red, and the protospacer-adjacent motif (PAM) sequence is labeled in purple. The different sgRNAs are marked as F1, F2, R1 and R2, respectively. The restriction sites at the target regions are underlined. Restriction enzymes used for RFLP analysis are shown. (B) Top: schematic diagram of the templates for in vitro transcription used to generate the Cas9 mRNA and sgRNA. Bottom: assessing the quality of sgRNAs and Cas9 mRNA by gel electrophoresis. Electrophoresis of sgRNAs yields a single band approximately 100 bp. Electrophoresis of Cas9 mRNA yields 2–3 bands due to persistent secondary structure. The quality of mRNA is good if discrete bands are visible.
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f1: Design and construction of CRISPR.(A) Schematic of the Cas9/sgRNA-targeting sites in pig mitfloci. Exons are shown as black boxes. The sgRNA-targeting sequence is labeled in red, and the protospacer-adjacent motif (PAM) sequence is labeled in purple. The different sgRNAs are marked as F1, F2, R1 and R2, respectively. The restriction sites at the target regions are underlined. Restriction enzymes used for RFLP analysis are shown. (B) Top: schematic diagram of the templates for in vitro transcription used to generate the Cas9 mRNA and sgRNA. Bottom: assessing the quality of sgRNAs and Cas9 mRNA by gel electrophoresis. Electrophoresis of sgRNAs yields a single band approximately 100 bp. Electrophoresis of Cas9 mRNA yields 2–3 bands due to persistent secondary structure. The quality of mRNA is good if discrete bands are visible.

Mentions: MITF protein is a master regulator of melanocyte development and an important oncogene in melanoma33. Mutations in the human mitf gene have been found in patients with the hypopigmentation and deafness syndromes, Waardenburg (WS) and Tietz (TS)34. Recently, numerous pig models of human diseases have been developed using gene targeting approach owing to pig sharing more physiological similarities with humans. It prompts us to generate mitf genes knockout pigs to model human WS and TS syndromes. We designed four different sgRNAs (F1, F2, R1 and R2) that target 47 bp regions of exon 8 of the pig mitf gene (Fig. 1A), which is a part of the basic helix-loop-helix leucine zipper (bHLH-Zip) domain sequence and is essential for MITF DNA-binding activity35. The sgRNA target sequence (20 nt) did not cross-react with any other sites in the pig genome and was followed by an NGG protospacer adjacent motif (PAM), which is necessary for Cas9 cleavage.


Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs.

Wang X, Zhou J, Cao C, Huang J, Hai T, Wang Y, Zheng Q, Zhang H, Qin G, Miao X, Wang H, Cao S, Zhou Q, Zhao J - Sci Rep (2015)

Design and construction of CRISPR.(A) Schematic of the Cas9/sgRNA-targeting sites in pig mitfloci. Exons are shown as black boxes. The sgRNA-targeting sequence is labeled in red, and the protospacer-adjacent motif (PAM) sequence is labeled in purple. The different sgRNAs are marked as F1, F2, R1 and R2, respectively. The restriction sites at the target regions are underlined. Restriction enzymes used for RFLP analysis are shown. (B) Top: schematic diagram of the templates for in vitro transcription used to generate the Cas9 mRNA and sgRNA. Bottom: assessing the quality of sgRNAs and Cas9 mRNA by gel electrophoresis. Electrophoresis of sgRNAs yields a single band approximately 100 bp. Electrophoresis of Cas9 mRNA yields 2–3 bands due to persistent secondary structure. The quality of mRNA is good if discrete bands are visible.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543986&req=5

f1: Design and construction of CRISPR.(A) Schematic of the Cas9/sgRNA-targeting sites in pig mitfloci. Exons are shown as black boxes. The sgRNA-targeting sequence is labeled in red, and the protospacer-adjacent motif (PAM) sequence is labeled in purple. The different sgRNAs are marked as F1, F2, R1 and R2, respectively. The restriction sites at the target regions are underlined. Restriction enzymes used for RFLP analysis are shown. (B) Top: schematic diagram of the templates for in vitro transcription used to generate the Cas9 mRNA and sgRNA. Bottom: assessing the quality of sgRNAs and Cas9 mRNA by gel electrophoresis. Electrophoresis of sgRNAs yields a single band approximately 100 bp. Electrophoresis of Cas9 mRNA yields 2–3 bands due to persistent secondary structure. The quality of mRNA is good if discrete bands are visible.
Mentions: MITF protein is a master regulator of melanocyte development and an important oncogene in melanoma33. Mutations in the human mitf gene have been found in patients with the hypopigmentation and deafness syndromes, Waardenburg (WS) and Tietz (TS)34. Recently, numerous pig models of human diseases have been developed using gene targeting approach owing to pig sharing more physiological similarities with humans. It prompts us to generate mitf genes knockout pigs to model human WS and TS syndromes. We designed four different sgRNAs (F1, F2, R1 and R2) that target 47 bp regions of exon 8 of the pig mitf gene (Fig. 1A), which is a part of the basic helix-loop-helix leucine zipper (bHLH-Zip) domain sequence and is essential for MITF DNA-binding activity35. The sgRNA target sequence (20 nt) did not cross-react with any other sites in the pig genome and was followed by an NGG protospacer adjacent motif (PAM), which is necessary for Cas9 cleavage.

Bottom Line: Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks.The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%.Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, the uncertainties caused by wide variations in sgRNA activity impede the utility of this system in generating genetically modified pigs. Here, we described a single blastocyst genotyping system to provide a simple and rapid solution to evaluate and compare the sgRNA efficiency at inducing indel mutations for a given gene locus. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%. Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer. We further showed that direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Thus, our method considerably reduces the uncertainties and expands the practical possibilities of CRISPR/Cas9-mediated genome engineering in pigs.

No MeSH data available.