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

BMC Biotechnol

State 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 University, Chongqing, 400715, People's Republic of China.

Published: September 2021

AI Article Synopsis

  • CRISPR/Cas9 technology has advanced gene editing in silkworms, with the new DNA-free ribonucleoprotein delivery method offering significant advantages over traditional methods.
  • The study explored adding extra G residues to sgRNA sequences to bypass limitations imposed by the 5' GG motif required for effective gene targeting, leading to successful mutations despite mismatches.
  • Results showed that sgRNAs with 5' GG mismatches can still induce mutations efficiently, and these mutations are heritable, enhancing the CRISPR/Cas9 system's effectiveness in silkworm gene editing.

Article 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.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8454041PMC
http://dx.doi.org/10.1186/s12896-021-00714-6DOI Listing

Publication Analysis

Top Keywords

crispr/cas9 system
12
targetable sites
8
ribonucleoprotein-based crispr/cas9
8
system silkworm
8
sgrnas
5
expansion targetable
4
sites ribonucleoprotein-based
4
crispr/cas9
4
silkworm
4
silkworm bombyx
4

Similar Publications

Fibroblasts display complex functions associated with distinct gene expression profiles that influence matrix production and cell communications and the autonomy of tissue development and repair. Thrombospondin-2 (TSP-2), produced by fibroblasts, is a potent angiogenesis inhibitor and negatively associated with tissue repair. Single-cell (sc) sequencing analysis on WT and TSP2KO skin fibroblasts demonstrate distinct cell heterogeneity.

View Article and Find Full Text PDF

Potato ( ) is the third most important food crop in the world. Although the potato genome has been fully sequenced, functional genomics research of potato lags relative to other major food crops due primarily to the lack of a model experimental potato line. Here, we present a diploid potato line, 'Jan', which possesses all essential characteristics for facile functional genomics studies.

View Article and Find Full Text PDF

Successful transgenesis in model organisms has dramatically helped us understand gene function, regulation, genetic networks, and potential applications. Here, we introduce the universal single-copy knock-in system (Universal SKI System or U-SKI), designed for inserting any transgene by CRISPR/Cas9 in the genome. The Universal SKI System takes advantage of a plasmid (pSKI), which can also be used for extrachromosomal arrays, to facilitate the insertion of a transgene at specific safe harbor loci on each autosomal chromosome.

View Article and Find Full Text PDF

CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) has revolutionized gene editing tools and paved the way for innovations in medical research for disease diagnosis and treatment. However, better specificity and efficient delivery of this gene machinery make it challenging to successfully edit genes for treating various diseases. This is mainly due to cellular barriers, instability in biological environments, and various off-target effects that prohibit safe and efficient delivery under in vivo conditions.

View Article and Find Full Text PDF

In the post-COVID-19 era, drug-resistant bacterial infections emerge as one of major death causes, where multidrug-resistant Acinetobacter baumannii (MRAB) and drug-resistant Pseudomonas aeruginosa (DRPA) represent primary pathogens. However, the classical antibiotic strategy currently faces the bottleneck of drug resistance. We develop an antimicrobial strategy that applies the selective delivery of CRISPR/Cas9 plasmids to pathogens with biomimetic cationic hybrid vesicles (BCVs), irrelevant to bacterial drug resistance.

View Article and Find Full Text PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!