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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.


Characterization of genome editing.(A) Editing frequency of transgenic plants. The number on the bar indicates the percentage of each genotype in all edited plants. (B) Editing types. The number on the bar indicates the percentage of each editing type in all edited sites. In (insertion), Del (deletion), Sub (substitution) and Com (complex). (C) Inversion of the DNA fragment. Shown are the amplified upstream and downstream junctions as well as their sequences. Deleted bases are indicated by dashes. Arrow indicates the predicted cleavage site. The red 3-base sequence indicates the PAM motif. Guide 15/16 sequences are indicated. (D) Comparison of editing rate between constructs with different number of sgRNA cassettes.
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f3: Characterization of genome editing.(A) Editing frequency of transgenic plants. The number on the bar indicates the percentage of each genotype in all edited plants. (B) Editing types. The number on the bar indicates the percentage of each editing type in all edited sites. In (insertion), Del (deletion), Sub (substitution) and Com (complex). (C) Inversion of the DNA fragment. Shown are the amplified upstream and downstream junctions as well as their sequences. Deleted bases are indicated by dashes. Arrow indicates the predicted cleavage site. The red 3-base sequence indicates the PAM motif. Guide 15/16 sequences are indicated. (D) Comparison of editing rate between constructs with different number of sgRNA cassettes.

Mentions: To detect the Cas9-sgRNA-mediated precise genome editing, T0 transgenic plants were used for sequencing analysis of desired target sites. PCR products covering predicted target sites were directly used for sequencing analysis. As expected, sgRNA 1 to 21 caused genome editing in the predicted target sites. In 371 sequenced plants (except for sgRNA22 and sgRNA23), 305 plants (82.2%) had mutations, which can be classified into homozygous (28.1%), heterozygous (14.2%), biallelic (56.7%) and chimeric (1.0%) mutations (Fig. 3A). In contrast to the 21 sgRNAs (sgRNA1 to sgRNA21) with the high editing rate, no target mutation was detected in all T0 transgenic plants containing sgRNA22 and sgRNA23 (20% G/C content) which do not comply with the criteria. sgRNA249 is a confirmed inefficient sgRNA which contains 14 CBPs and does not agree with the criteria (Supplemental Table 3). These evidence suggested that the criteria are reliable for selection of efficient sgRNAs.


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

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

Characterization of genome editing.(A) Editing frequency of transgenic plants. The number on the bar indicates the percentage of each genotype in all edited plants. (B) Editing types. The number on the bar indicates the percentage of each editing type in all edited sites. In (insertion), Del (deletion), Sub (substitution) and Com (complex). (C) Inversion of the DNA fragment. Shown are the amplified upstream and downstream junctions as well as their sequences. Deleted bases are indicated by dashes. Arrow indicates the predicted cleavage site. The red 3-base sequence indicates the PAM motif. Guide 15/16 sequences are indicated. (D) Comparison of editing rate between constructs with different number of sgRNA cassettes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Characterization of genome editing.(A) Editing frequency of transgenic plants. The number on the bar indicates the percentage of each genotype in all edited plants. (B) Editing types. The number on the bar indicates the percentage of each editing type in all edited sites. In (insertion), Del (deletion), Sub (substitution) and Com (complex). (C) Inversion of the DNA fragment. Shown are the amplified upstream and downstream junctions as well as their sequences. Deleted bases are indicated by dashes. Arrow indicates the predicted cleavage site. The red 3-base sequence indicates the PAM motif. Guide 15/16 sequences are indicated. (D) Comparison of editing rate between constructs with different number of sgRNA cassettes.
Mentions: To detect the Cas9-sgRNA-mediated precise genome editing, T0 transgenic plants were used for sequencing analysis of desired target sites. PCR products covering predicted target sites were directly used for sequencing analysis. As expected, sgRNA 1 to 21 caused genome editing in the predicted target sites. In 371 sequenced plants (except for sgRNA22 and sgRNA23), 305 plants (82.2%) had mutations, which can be classified into homozygous (28.1%), heterozygous (14.2%), biallelic (56.7%) and chimeric (1.0%) mutations (Fig. 3A). In contrast to the 21 sgRNAs (sgRNA1 to sgRNA21) with the high editing rate, no target mutation was detected in all T0 transgenic plants containing sgRNA22 and sgRNA23 (20% G/C content) which do not comply with the criteria. sgRNA249 is a confirmed inefficient sgRNA which contains 14 CBPs and does not agree with the criteria (Supplemental Table 3). These evidence suggested that the criteria are reliable for selection of efficient sgRNAs.

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.