Enhancements of the CRISPR-Cas System in the Silkworm Bombyx mori.

IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY
CRISPR Journal Pub Date : 2025-04-01 Epub Date: 2025-03-27 DOI:10.1089/crispr.2024.0089
Takuya Tsubota, Yoko Takasu, Naoyuki Yonemura, Hideki Sezutsu
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引用次数: 0

Abstract

The silkworm (Bombyx mori) is a lepidopteran model insect that has been utilized for basic research and industrial applications. In this species, transcription activator-like effector nucleases (TALENs) have been found to function efficiently, and we previously developed a TALEN-mediated genome editing system for knockout and knock-in experiments using plasmids and single-stranded oligodeoxynucleotides (ssODNs) as donors. By contrast, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing, especially for gene integration, remains limited. In this study, we attempted to improve CRISPR-Cas systems to expand the utility of genome editing in the silkworm. Codon optimization of Cas9 improved genome editing efficiency, and single-guide RNA utilization also resulted in a higher genome editing efficiency than crRNA/tracrRNA when Cas9 messenger RNA (mRNA) was used. CRISPR-Cas12a-mediated genome editing and targeted sequence integration using ssODNs were both successfully performed. Overall, our study provides a robust technical platform that can facilitate basic and applied silkworm studies.

家蚕CRISPR-Cas系统的增强。
家蚕(Bombyx mori)是鳞翅目模式昆虫,已被用于基础研究和工业应用。在这个物种中,转录激活因子样效应核酸酶(TALENs)已经被发现有效地发挥作用,我们之前开发了一个talen介导的基因组编辑系统,用于敲除和敲入实验,使用质粒和单链寡脱氧核苷酸(ssODNs)作为供体。相比之下,聚集规律间隔短回文重复(CRISPR)-CRISPR相关蛋白9 (Cas9)介导的基因组编辑,特别是基因整合,仍然有限。在本研究中,我们试图改进CRISPR-Cas系统,以扩大基因组编辑在家蚕中的应用。Cas9的密码子优化提高了基因组编辑效率,当使用Cas9信使RNA (mRNA)时,单导RNA的使用也使基因组编辑效率高于crRNA/tracrRNA。crispr - cas12a介导的基因组编辑和使用ssODNs的靶向序列整合都成功完成。总的来说,我们的研究提供了一个强大的技术平台,可以促进基础和应用蚕的研究。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CRISPR Journal
CRISPR Journal Biochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
6.30
自引率
2.70%
发文量
76
期刊介绍: In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR. Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.
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