{"title":"通过 CRISPR/Cas9 工程培育携带鳄鱼鞘氨醇基因的环保抗病峡鲶(Ictalurus punctatus)基因","authors":"","doi":"10.1016/j.eng.2023.12.005","DOIUrl":null,"url":null,"abstract":"<div><p>As a precise and versatile tool for genome manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) platform holds promise for modifying fish traits of interest. With the aim of reducing transgene introgression and controlling reproduction, upscaled disease resistance and reproductive intervention in catfish species have been studied to lower the potential environmental risks of the introgression of escapees as transgenic animals. Taking advantage of the CRISPR/Cas9-mediated system, we succeeded in integrating the cathelicidin gene (<em>As</em>-<em>Cath</em>) from an alligator (<em>Alligator sinensis</em>) into the target luteinizing hormone (<em>lh</em>) locus of channel catfish (<em>Ictalurus punctatus</em>) using two delivery systems assisted by double-stranded DNA (dsDNA) and single-stranded oligodeoxynucleotides (ssODNs), respectively. In this study, high knock in (KI) efficiency (22.38%, 64/286) but low on-target events was achieved using the ssODN strategy, whereas adopting a dsDNA as the donor template led to an efficient on-target KI (10.80%, 23/213). The on-target KI of <em>As-Cath</em> was instrumental in establishing the <em>lh</em> knockout (LH<sup>–</sup>_As-Cath<sup>+</sup>) catfish line, which displayed heightened disease resistance and reduced fecundity compared with the wild-type (WT) sibling fish. Furthermore, administration of human chorionic gonadotropin (HCG) and luteinizing hormone-releasing hormone analogue (LHRHa) can restore the reproduction of the transgenic fish line. Overall, we replaced the <em>lh</em> gene with an alligator cathelicidin transgene and then administered hormone therapy to move towards complete reproductive control of disease-resistant transgenic catfish in an environmentally responsible manner. This strategy not only effectively improves consumer-valued traits but also guards against unwanted introgression, providing a breakthrough in aquaculture genetics to confine fish reproduction and prevent the establishment of transgenic or domestic genotypes in the natural environment.</p></div>","PeriodicalId":11783,"journal":{"name":"Engineering","volume":null,"pages":null},"PeriodicalIF":10.1000,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2095809924000420/pdfft?md5=44370087181758a7e38b5f9f08adfffa&pid=1-s2.0-S2095809924000420-main.pdf","citationCount":"0","resultStr":"{\"title\":\"Generation of Eco-Friendly and Disease-Resistant Channel Catfish (Ictalurus punctatus) Harboring the Alligator Cathelicidin Gene via CRISPR/Cas9 Engineering\",\"authors\":\"\",\"doi\":\"10.1016/j.eng.2023.12.005\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>As a precise and versatile tool for genome manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) platform holds promise for modifying fish traits of interest. With the aim of reducing transgene introgression and controlling reproduction, upscaled disease resistance and reproductive intervention in catfish species have been studied to lower the potential environmental risks of the introgression of escapees as transgenic animals. Taking advantage of the CRISPR/Cas9-mediated system, we succeeded in integrating the cathelicidin gene (<em>As</em>-<em>Cath</em>) from an alligator (<em>Alligator sinensis</em>) into the target luteinizing hormone (<em>lh</em>) locus of channel catfish (<em>Ictalurus punctatus</em>) using two delivery systems assisted by double-stranded DNA (dsDNA) and single-stranded oligodeoxynucleotides (ssODNs), respectively. In this study, high knock in (KI) efficiency (22.38%, 64/286) but low on-target events was achieved using the ssODN strategy, whereas adopting a dsDNA as the donor template led to an efficient on-target KI (10.80%, 23/213). The on-target KI of <em>As-Cath</em> was instrumental in establishing the <em>lh</em> knockout (LH<sup>–</sup>_As-Cath<sup>+</sup>) catfish line, which displayed heightened disease resistance and reduced fecundity compared with the wild-type (WT) sibling fish. Furthermore, administration of human chorionic gonadotropin (HCG) and luteinizing hormone-releasing hormone analogue (LHRHa) can restore the reproduction of the transgenic fish line. Overall, we replaced the <em>lh</em> gene with an alligator cathelicidin transgene and then administered hormone therapy to move towards complete reproductive control of disease-resistant transgenic catfish in an environmentally responsible manner. This strategy not only effectively improves consumer-valued traits but also guards against unwanted introgression, providing a breakthrough in aquaculture genetics to confine fish reproduction and prevent the establishment of transgenic or domestic genotypes in the natural environment.</p></div>\",\"PeriodicalId\":11783,\"journal\":{\"name\":\"Engineering\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":10.1000,\"publicationDate\":\"2024-08-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S2095809924000420/pdfft?md5=44370087181758a7e38b5f9f08adfffa&pid=1-s2.0-S2095809924000420-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Engineering\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S2095809924000420\",\"RegionNum\":1,\"RegionCategory\":\"工程技术\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"ENGINEERING, MULTIDISCIPLINARY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Engineering","FirstCategoryId":"5","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2095809924000420","RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ENGINEERING, MULTIDISCIPLINARY","Score":null,"Total":0}
引用次数: 0
摘要
作为一种精确而多用途的基因组操作工具,簇状规则间隔短回文重复序列(CRISPR)/CRISPR相关蛋白9(Cas9)平台有望改变鱼类的相关性状。为了减少转基因导入和控制繁殖,对鲶鱼物种的抗病性和繁殖干预进行了升级研究,以降低转基因动物逃逸者导入的潜在环境风险。利用CRISPR/Cas9介导系统的优势,我们分别采用双链DNA(dsDNA)和单链寡核苷酸(ssODNs)两种传递系统,成功地将短吻鳄(Alligator sinensis)的cathelicidin基因(As-Cath)整合到了斑点叉尾鮰(Ictalurus punctatus)的黄体生成素(lh)基因座上。在这项研究中,使用ssODN策略实现了较高的敲入(KI)效率(22.38%,64/286),但靶上事件较少,而采用dsDNA作为供体模板则实现了高效的靶上KI(10.80%,23/213)。As-Cath 的靶上 KI 在建立 lh 基因敲除(LH-_As-Cath+)鲶鱼品系中发挥了重要作用,与野生型(WT)同胞鱼相比,该品系表现出更强的抗病性,但繁殖力降低。此外,注射人绒毛膜促性腺激素(HCG)和促黄体生成素释放激素类似物(LHRHa)可恢复转基因鱼系的繁殖能力。总之,我们用鳄鱼鞘磷脂转基因取代 lh 基因,然后施用激素疗法,以无害环境的方式完全控制了抗病转基因鲶鱼的繁殖。这种策略不仅能有效改善消费者看重的性状,还能防止不必要的基因导入,在水产养殖遗传学方面实现了突破,限制了鱼类的繁殖,防止了转基因或家养基因型在自然环境中的建立。
Generation of Eco-Friendly and Disease-Resistant Channel Catfish (Ictalurus punctatus) Harboring the Alligator Cathelicidin Gene via CRISPR/Cas9 Engineering
As a precise and versatile tool for genome manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) platform holds promise for modifying fish traits of interest. With the aim of reducing transgene introgression and controlling reproduction, upscaled disease resistance and reproductive intervention in catfish species have been studied to lower the potential environmental risks of the introgression of escapees as transgenic animals. Taking advantage of the CRISPR/Cas9-mediated system, we succeeded in integrating the cathelicidin gene (As-Cath) from an alligator (Alligator sinensis) into the target luteinizing hormone (lh) locus of channel catfish (Ictalurus punctatus) using two delivery systems assisted by double-stranded DNA (dsDNA) and single-stranded oligodeoxynucleotides (ssODNs), respectively. In this study, high knock in (KI) efficiency (22.38%, 64/286) but low on-target events was achieved using the ssODN strategy, whereas adopting a dsDNA as the donor template led to an efficient on-target KI (10.80%, 23/213). The on-target KI of As-Cath was instrumental in establishing the lh knockout (LH–_As-Cath+) catfish line, which displayed heightened disease resistance and reduced fecundity compared with the wild-type (WT) sibling fish. Furthermore, administration of human chorionic gonadotropin (HCG) and luteinizing hormone-releasing hormone analogue (LHRHa) can restore the reproduction of the transgenic fish line. Overall, we replaced the lh gene with an alligator cathelicidin transgene and then administered hormone therapy to move towards complete reproductive control of disease-resistant transgenic catfish in an environmentally responsible manner. This strategy not only effectively improves consumer-valued traits but also guards against unwanted introgression, providing a breakthrough in aquaculture genetics to confine fish reproduction and prevent the establishment of transgenic or domestic genotypes in the natural environment.
期刊介绍:
Engineering, an international open-access journal initiated by the Chinese Academy of Engineering (CAE) in 2015, serves as a distinguished platform for disseminating cutting-edge advancements in engineering R&D, sharing major research outputs, and highlighting key achievements worldwide. The journal's objectives encompass reporting progress in engineering science, fostering discussions on hot topics, addressing areas of interest, challenges, and prospects in engineering development, while considering human and environmental well-being and ethics in engineering. It aims to inspire breakthroughs and innovations with profound economic and social significance, propelling them to advanced international standards and transforming them into a new productive force. Ultimately, this endeavor seeks to bring about positive changes globally, benefit humanity, and shape a new future.