Deep orange gene editing triggers temperature-sensitive lethal phenotypes in Ceratitis capitata

IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY BMC Biotechnology Pub Date : 2024-02-01 DOI:10.1186/s12896-024-00832-x
Germano Sollazzo, Katerina Nikolouli, Georgia Gouvi, Roswitha A. Aumann, Marc F. Schetelig, Kostas Bourtzis
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Abstract

The Mediterranean fruit fly, Ceratitis capitata, is a significant agricultural pest managed through area-wide integrated pest management (AW-IPM) including a sterile insect technique (SIT) component. Male-only releases increase the efficiency and cost-effectiveness of SIT programs, which can be achieved through the development of genetic sexing strains (GSS). The most successful GSS developed to date is the C. capitata VIENNA 8 GSS, constructed using classical genetic approaches and an irradiation-induced translocation with two selectable markers: the white pupae (wp) and temperature-sensitive lethal (tsl) genes. However, currently used methods for selecting suitable markers and inducing translocations are stochastic and non-specific, resulting in a laborious and time-consuming process. Recent efforts have focused on identifying the gene(s) and the causal mutation(s) for suitable phenotypes, such as wp and tsl, which could be used as selectable markers for developing a generic approach for constructing GSS. The wp gene was recently identified, and efforts have been initiated to identify the tsl gene. This study investigates Ceratitis capitata deep orange (Ccdor) as a tsl candidate gene and its potential to induce tsl phenotypes. An integrated approach based on cytogenetics, genomics, bioinformatics, and gene editing was used to characterize the Ccdor. Its location was confirmed on the right arm of chromosome 5 in the putative tsl genomic region. Knock-out of Ccdor using CRISPR/Cas9-NHEJ and targeting the fourth exon resulted in lethality at mid- and late-pupal stage, while the successful application of CRISPR HDR introducing a point mutation on the sixth exon resulted in the establishment of the desired strain and two additional strains (dor 12del and dor 51dup), all of them expressing tsl phenotypes and presenting no (or minimal) fitness cost when reared at 25 °C. One of the strains exhibited complete lethality when embryos were exposed at 36 °C. Gene editing of the deep orange gene in Ceratitis capitata resulted in the establishment of temperature-sensitive lethal mutant strains. The induced mutations did not significantly affect the rearing efficiency of the strains. As deep orange is a highly conserved gene, these data suggest that it can be considered a target for the development of tsl mutations which could potentially be used to develop novel genetic sexing strains in insect pests and disease vectors.
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深橙色基因编辑引发角膜炎的温度敏感致死表型
地中海果蝇(Ceratitis capitata)是一种重要的农业害虫,通过包括昆虫不育技术(SIT)在内的全区域害虫综合防治(AW-IPM)进行管理。只释放雄蝇可提高 SIT 计划的效率和成本效益,这可通过开发遗传性别品系(GSS)来实现。迄今为止最成功的 GSS 是 C. capitata VIENNA 8 GSS,它是利用经典遗传方法和辐照诱导易位法构建的,带有两个可选择标记:白蛹基因(wp)和温敏致死基因(tsl)。然而,目前使用的选择合适标记和诱导易位的方法具有随机性和非特异性,导致过程费时费力。最近的工作重点是确定合适表型(如 wp 和 tsl)的基因和致病突变,这些基因和突变可用作选择性标记,用于开发构建 GSS 的通用方法。最近确定了 wp 基因,并已开始努力确定 tsl 基因。本研究调查了作为tsl候选基因的角叉菜深橙(Ccdor)及其诱导tsl表型的潜力。研究采用了一种基于细胞遗传学、基因组学、生物信息学和基因编辑的综合方法来描述 Ccdor 的特征。它的位置被确认在推定的tsl基因组区域的5号染色体右臂上。利用 CRISPR/Cas9-NHEJ 并以第四外显子为靶基因敲除 Ccdor 后,在蛹的中期和晚期会导致致死,而成功应用 CRISPR HDR 在第六外显子上引入点突变后,建立了所需的品系和另外两个品系(dor 12del 和 dor 51dup),它们都表达了tsl表型,并在 25 °C饲养时没有(或只有极少)适应性代价。当胚胎暴露于 36 °C时,其中一个品系表现出完全致死。对钝顶角蛙的深橙色基因进行基因编辑后,产生了对温度敏感的致死突变株。诱导的突变并没有明显影响菌株的饲养效率。由于深橙色基因是一个高度保守的基因,这些数据表明它可被视为开发tsl突变的目标,有可能用于开发昆虫害虫和疾病媒介的新型基因性别株系。
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
BMC Biotechnology
BMC Biotechnology 工程技术-生物工程与应用微生物
CiteScore
6.60
自引率
0.00%
发文量
34
审稿时长
2 months
期刊介绍: BMC Biotechnology is an open access, peer-reviewed journal that considers articles on the manipulation of biological macromolecules or organisms for use in experimental procedures, cellular and tissue engineering or in the pharmaceutical, agricultural biotechnology and allied industries.
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