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Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing.

Liang G, Zhang H, Lou D, Yu D - Sci Rep (2016)

Bottom Line: As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed.Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency.This work provides a convenient approach to select efficient sgRNAs for target editing.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Kunming, Yunnan 650223, China.

ABSTRACT
The CRISPR/Cas9-sgRNA system has been developed to mediate genome editing and become a powerful tool for biological research. Employing the CRISPR/Cas9-sgRNA system for genome editing and manipulation has accelerated research and expanded researchers' ability to generate genetic models. However, the method evaluating the efficiency of sgRNAs is lacking in plants. Based on the nucleotide compositions and secondary structures of sgRNAs which have been experimentally validated in plants, we instituted criteria to design efficient sgRNAs. To facilitate the assembly of multiple sgRNA cassettes, we also developed a new strategy to rapidly construct CRISPR/Cas9-sgRNA system for multiplex editing in plants. In theory, up to ten single guide RNA (sgRNA) cassettes can be simultaneously assembled into the final binary vectors. As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed. Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency. This work provides a convenient approach to select efficient sgRNAs for target editing.

No MeSH data available.


Construction strategy of CRISPR/Cas9-sgRNA system.(A) Generation of an intact sgRNA cassette. Guide sequence (blue bar) containing chimeric primers were used to perform over-lapping PCR to generate an sgRNA cassette. The arrow-circle indicates the public primer with specific restriction enzyme sites. (B) Multiple clone sites of the intermediate vector pSAK2. 11 regular restriction enzyme sites are included, which can be used for assembly of up to 10 sgRNA cassettes in theory. (C) Structures of the binary vectors based on the pCAMBIA1300 backbone. HPT, Bar, and NPT II encode hygromycin B phosphotransferase, PPT acetyltransferase and neomycin phosphotransferase II, respectively. Two restriction enzyme sites, Spe I/Asc I, were provided for the entry of multiple sgRNA cassettes.
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f2: Construction strategy of CRISPR/Cas9-sgRNA system.(A) Generation of an intact sgRNA cassette. Guide sequence (blue bar) containing chimeric primers were used to perform over-lapping PCR to generate an sgRNA cassette. The arrow-circle indicates the public primer with specific restriction enzyme sites. (B) Multiple clone sites of the intermediate vector pSAK2. 11 regular restriction enzyme sites are included, which can be used for assembly of up to 10 sgRNA cassettes in theory. (C) Structures of the binary vectors based on the pCAMBIA1300 backbone. HPT, Bar, and NPT II encode hygromycin B phosphotransferase, PPT acetyltransferase and neomycin phosphotransferase II, respectively. Two restriction enzyme sites, Spe I/Asc I, were provided for the entry of multiple sgRNA cassettes.

Mentions: The first step is to construct sgRNA expression cassettes containing the guide sequences. The guide sequences can be easily integrated into sgRNA expression cassettes by overlapping PCR with guide sequence-containing chimeric primers (Fig. 2A). At the same time, ten pairs of public primers, which can pair with the 5′-terminal of promoters and the 3′-terminal of sgRNA and contain specific restriction enzyme sites, are available (Supplemental Table 2). The public primers of interest should be selected according to the desired order of sgRNA expression cassettes. After two rounds of PCR, an intact sgRNA expression cassette with two different restriction enzyme sites in the 5′ and 3′ ends can be produced (Fig. 2A). The second step is to clone different sgRNA expression cassettes into the intermediate vector pSAK2 (Fig. 2B). The third step is to digest pSAK2-sgRNA(s) by Spe I/Asc I and then ligate the sgRNA(s) into the final binary vectors (Fig. 2C; Supplemental Fig. 2).


Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing.

Liang G, Zhang H, Lou D, Yu D - Sci Rep (2016)

Construction strategy of CRISPR/Cas9-sgRNA system.(A) Generation of an intact sgRNA cassette. Guide sequence (blue bar) containing chimeric primers were used to perform over-lapping PCR to generate an sgRNA cassette. The arrow-circle indicates the public primer with specific restriction enzyme sites. (B) Multiple clone sites of the intermediate vector pSAK2. 11 regular restriction enzyme sites are included, which can be used for assembly of up to 10 sgRNA cassettes in theory. (C) Structures of the binary vectors based on the pCAMBIA1300 backbone. HPT, Bar, and NPT II encode hygromycin B phosphotransferase, PPT acetyltransferase and neomycin phosphotransferase II, respectively. Two restriction enzyme sites, Spe I/Asc I, were provided for the entry of multiple sgRNA cassettes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4759811&req=5

f2: Construction strategy of CRISPR/Cas9-sgRNA system.(A) Generation of an intact sgRNA cassette. Guide sequence (blue bar) containing chimeric primers were used to perform over-lapping PCR to generate an sgRNA cassette. The arrow-circle indicates the public primer with specific restriction enzyme sites. (B) Multiple clone sites of the intermediate vector pSAK2. 11 regular restriction enzyme sites are included, which can be used for assembly of up to 10 sgRNA cassettes in theory. (C) Structures of the binary vectors based on the pCAMBIA1300 backbone. HPT, Bar, and NPT II encode hygromycin B phosphotransferase, PPT acetyltransferase and neomycin phosphotransferase II, respectively. Two restriction enzyme sites, Spe I/Asc I, were provided for the entry of multiple sgRNA cassettes.
Mentions: The first step is to construct sgRNA expression cassettes containing the guide sequences. The guide sequences can be easily integrated into sgRNA expression cassettes by overlapping PCR with guide sequence-containing chimeric primers (Fig. 2A). At the same time, ten pairs of public primers, which can pair with the 5′-terminal of promoters and the 3′-terminal of sgRNA and contain specific restriction enzyme sites, are available (Supplemental Table 2). The public primers of interest should be selected according to the desired order of sgRNA expression cassettes. After two rounds of PCR, an intact sgRNA expression cassette with two different restriction enzyme sites in the 5′ and 3′ ends can be produced (Fig. 2A). The second step is to clone different sgRNA expression cassettes into the intermediate vector pSAK2 (Fig. 2B). The third step is to digest pSAK2-sgRNA(s) by Spe I/Asc I and then ligate the sgRNA(s) into the final binary vectors (Fig. 2C; Supplemental Fig. 2).

Bottom Line: As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed.Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency.This work provides a convenient approach to select efficient sgRNAs for target editing.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Kunming, Yunnan 650223, China.

ABSTRACT
The CRISPR/Cas9-sgRNA system has been developed to mediate genome editing and become a powerful tool for biological research. Employing the CRISPR/Cas9-sgRNA system for genome editing and manipulation has accelerated research and expanded researchers' ability to generate genetic models. However, the method evaluating the efficiency of sgRNAs is lacking in plants. Based on the nucleotide compositions and secondary structures of sgRNAs which have been experimentally validated in plants, we instituted criteria to design efficient sgRNAs. To facilitate the assembly of multiple sgRNA cassettes, we also developed a new strategy to rapidly construct CRISPR/Cas9-sgRNA system for multiplex editing in plants. In theory, up to ten single guide RNA (sgRNA) cassettes can be simultaneously assembled into the final binary vectors. As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed. Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency. This work provides a convenient approach to select efficient sgRNAs for target editing.

No MeSH data available.