Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori
Abstract 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 a...
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doaj-0478f5323894419d84b56370af38f8452021-09-26T11:41:22ZengBMCBMC Biotechnology1472-67502021-09-012111910.1186/s12896-021-00714-6Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx moriYun-long Zou0Ai-jun Ye1Shuo Liu2Wen-tao Wu3Li-feng Xu4Fang-yin Dai5Xiao-ling Tong6State Key Laboratory of Silkworm Genome Biology; Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs; College of Sericulture, Textile and Biomass Sciences, Southwest UniversityState Key Laboratory of Silkworm Genome Biology; Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs; College of Sericulture, Textile and Biomass Sciences, Southwest UniversityState Key Laboratory of Silkworm Genome Biology; Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs; College of Sericulture, Textile and Biomass Sciences, Southwest UniversityState Key Laboratory of Silkworm Genome Biology; Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs; College of Sericulture, Textile and Biomass Sciences, Southwest UniversityState Key Laboratory of Silkworm Genome Biology; Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs; College of Sericulture, Textile and Biomass Sciences, Southwest UniversityState Key Laboratory of Silkworm Genome Biology; Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs; College of Sericulture, Textile and Biomass Sciences, Southwest UniversityState Key Laboratory of Silkworm Genome Biology; Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs; College of Sericulture, Textile and Biomass Sciences, Southwest UniversityAbstract 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.https://doi.org/10.1186/s12896-021-00714-6CRISPR/Cas9RibonucleoproteinSilkwormTarget repertoireBmBLOS2BmGR66 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yun-long Zou Ai-jun Ye Shuo Liu Wen-tao Wu Li-feng Xu Fang-yin Dai Xiao-ling Tong |
spellingShingle |
Yun-long Zou Ai-jun Ye Shuo Liu Wen-tao Wu Li-feng Xu Fang-yin Dai Xiao-ling Tong Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori BMC Biotechnology CRISPR/Cas9 Ribonucleoprotein Silkworm Target repertoire BmBLOS2 BmGR66 |
author_facet |
Yun-long Zou Ai-jun Ye Shuo Liu Wen-tao Wu Li-feng Xu Fang-yin Dai Xiao-ling Tong |
author_sort |
Yun-long Zou |
title |
Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori |
title_short |
Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori |
title_full |
Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori |
title_fullStr |
Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori |
title_full_unstemmed |
Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori |
title_sort |
expansion of targetable sites for the ribonucleoprotein-based crispr/cas9 system in the silkworm bombyx mori |
publisher |
BMC |
series |
BMC Biotechnology |
issn |
1472-6750 |
publishDate |
2021-09-01 |
description |
Abstract 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. |
topic |
CRISPR/Cas9 Ribonucleoprotein Silkworm Target repertoire BmBLOS2 BmGR66 |
url |
https://doi.org/10.1186/s12896-021-00714-6 |
work_keys_str_mv |
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