Enhanced <i>Bombyx</i> genome editing via Cas9 ribonucleoprotein injection

Lee, Jia; Ma, Sanyuan; Zhang, Tong; Xing, Weiqing; Liu, Yue; Li, Yufeng; Chen, Xiao‐Xu; Xia, Qingyou

doi:10.1111/1744-7917.12633

Cited by 5 publications

(8 citation statements)

References 16 publications

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“…The delivery of CRISPR/Cas9 system into silkworms has been accomplished using the binary transgenic method [3,[5][6][7] and the Cas9 mRNA-or protein-based methods [4,10]. In the binary method, the RNA polymerase type III promoter U6 is widely exploited to express sgRNA in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…However, when the binary transgene-based method is used to deliver CRISPR/Cas9, every new genomic target requires a new transgenic line to express speci c sgRNAs, limiting the exibility of the system. Therefore, the DNA-free RNP-based CRISPR/Cas9 system has become increasingly popular in silkworms [10]. In this method, sgRNAs are synthesized in vitro, typically from a T7 promoter because of its high e ciency.…”

Section: Discussionmentioning

confidence: 99%

“…Protocols using the Cas9 protein are more effective than those using Cas9 mRNA because the protein acts immediately following injection without a translational delay [9,10]. In the Cas9 ribonucleoprotein (RNP)-based method, the T7 promoter is often chosen for in vitro transcription of the sgRNA [10]. However, the T7 promoter requires a GG dinucleotide at the RNA transcriptional start site for e cient transcription.…”

Section: Introductionmentioning

confidence: 99%

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Expansion of Targetable Sites for the Ribonucleoprotein-Based CRISPR/Cas9 System in the Silkworm Bombyx Mori

Zou

Liu

et al. 2021

Preprint

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Background:With the emergence of CRISPR/Cas9 technology, multiple gene editing procedures became available for the silkworm. Although binary transgene-based methods have been widely used to generate mutants, delivery of the CRISPR/Cas9 system via DNA-free ribonucleoproteins offers several advantages. However, the T7 promoter that is widely used in the ribonucleoprotein-based method for production of sgRNAs in vitro requires a 5’ GG motif for efficient initiation. The resulting transcripts bear a 5’ GG motif, which significantly constrains the number of targetable sites in the silkworm genome. Results:In this study, we used the T7 promoter to add two supernumerary G residues to the 5’ end of conventional (perfectly matched) 20-nucleotide sgRNA targeting sequences. We then asked if sgRNAs with this structure can generate mutations even if the genomic target does not contain corresponding GG residues. As expected, 5’ GG mismatches depress the mutagenic activity of sgRNAs, and a single 5’ G mismatch has a relatively minor effect. However, tests involving six sgRNAs targeting two genes show that the mismatches do not eliminate mutagenesis in vivo, and the efficiencies remain at useable levels. One sgRNA with a 5’ GG mismatch at its target performed mutagenesis more efficiently than a conventional sgRNA with 5’ matched GG residues at a second target within the same gene. Mutations generated by sgRNAs with 5’ GG mismatches are also heritable. We successfully obtained null mutants with detectable phenotypes from sib-mated mosaics after one generation. Conclusions: In summary, our method improves the utility and flexibility of the ribonucleoprotein-based CRISPR/Cas9 system in silkworm.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Expansion of Targetable Sites for the Ribonucleoprotein-Based CRISPR/Cas9 System in the Silkworm Bombyx Mori

Zou

Liu

et al. 2021

Preprint

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“…The preparation of Cas9 protein and sgRNAs was performed according to the procedure we reported previously with minor modifications. 25 The plasmid of Cas9 was subcloned into a pET28a prokaryotic expression vector encoding an N-terminal 6 • His-fusion tag and was verified by Sanger sequencing. The expression vector was transformed into the Escherichia coli strain BL21 (DE3).…”

Section: Cas9 and Sgrnasmentioning

confidence: 99%

Deep Sequencing Reveals the Comprehensive CRISPR-Cas9 Editing Spectrum in Bombyx mori

Wang

Chen

et al. 2021

The CRISPR Journal

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Application of the clustered regularly interspaced short palindromic repeats associated 9 (CRISPR-Cas9) technology has revolutionized biology by greatly enhancing the ability to introduce mutations into DNA for research and prospective therapeutic purposes. However, the understanding of Cas9 editing outcomes is still limited. Previously, it was considered that Cas9 introduces stochastic insertions or deletions (indels) at the target site. In the current study, we performed in vivo multiplex editing, deep sequencing, and comprehensive analysis of its editing outcomes in Bombyx mori (B. mori). A total of 31161 editing events from 9 single-guide RNA (sgRNA) sites in 16 individuals were generated and analyzed, and we found that Cas9 introduces mutations with some regularity rather than via stochastic indels. The editing efficiency varies with sgRNA sequences, individuals, and orientation. Small deletions account for the vast majority of mutated sequences, followed by a small fraction of substitutions and insertions. The most likely mutations are deletions between two microhomologous sequences or singlebase deletions at the cleavage site in the absence of microhomologous pairs. Insertions are formed by diverse mechanisms, including direct acquisition of free genomic fragments, duplication of broken ends, replication of adjacent sequences, or random addition of free nucleotides. The above results indicate that the Cas9 editing spectrum is reproducible and predictable. Thus, our findings enable a deeper understanding of Cas9-mediated mutagenesis and better design of genome editing experiments, as well as elucidate the DNA double-strand break repair processes in B. mori.

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“…To overcome the drawbacks of the binary transgenic system, researchers have developed two DNA-free methods using Cas9 mRNA and Cas9 protein. Protocols using the Cas9 protein are more effective than those using Cas9 mRNA because the protein acts immediately following injection without a translational delay [ 9 , 10 ]. In the Cas9 ribonucleoprotein (RNP)-based method, the T7 promoter is often chosen for in vitro transcription of the sgRNA [ 10 ].…”

Section: Introductionmentioning

confidence: 99%

Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori

Zou

Liu

et al. 2021

BMC Biotechnol

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Background With the emergence of CRISPR/Cas9 technology, multiple gene editing procedures became available for the silkworm. Although binary transgene-based methods have been widely used to generate mutants, delivery of the CRISPR/Cas9 system via DNA-free ribonucleoproteins offers several advantages. However, the T7 promoter that is widely used in the ribonucleoprotein-based method for production of sgRNAs in vitro requires a 5′ GG motif for efficient initiation. The resulting transcripts bear a 5′ GG motif, which significantly constrains the number of targetable sites in the silkworm genome. Results In this study, we used the T7 promoter to add two supernumerary G residues to the 5′ end of conventional (perfectly matched) 20-nucleotide sgRNA targeting sequences. We then asked if sgRNAs with this structure can generate mutations even if the genomic target does not contain corresponding GG residues. As expected, 5′ GG mismatches depress the mutagenic activity of sgRNAs, and a single 5′ G mismatch has a relatively minor effect. However, tests involving six sgRNAs targeting two genes show that the mismatches do not eliminate mutagenesis in vivo, and the efficiencies remain at useable levels. One sgRNA with a 5′ GG mismatch at its target performed mutagenesis more efficiently than a conventional sgRNA with 5′ matched GG residues at a second target within the same gene. Mutations generated by sgRNAs with 5′ GG mismatches are also heritable. We successfully obtained null mutants with detectable phenotypes from sib-mated mosaics after one generation. Conclusions In summary, our method improves the utility and flexibility of the ribonucleoprotein-based CRISPR/Cas9 system in silkworm.

show abstract

Enhanced Bombyx genome editing via Cas9 ribonucleoprotein injection

Cited by 5 publications

References 16 publications

Expansion of Targetable Sites for the Ribonucleoprotein-Based CRISPR/Cas9 System in the Silkworm Bombyx Mori

Expansion of Targetable Sites for the Ribonucleoprotein-Based CRISPR/Cas9 System in the Silkworm Bombyx Mori

Deep Sequencing Reveals the Comprehensive CRISPR-Cas9 Editing Spectrum in Bombyx mori

Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori

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