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 gain complete reproductive control of disease-resistant transgenic catfish in an environmentally sound 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.

作为一种精确且多功能的基因组操作工具，成簇的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。为了减少转基因渗入和控制繁殖，对鲶鱼物种的抗病性和繁殖干预进行了研究，以降低逃逸者作为转基因动物渗入的潜在环境风险。利用 CRISPR/Cas9 介导的系统，我们使用两种递送系统成功地将扬子鳄 ( Alligator sinensis ) 的导管素基因 ( As - Cath ) 整合到斑点叉尾鮰 ( Ictalurus punctatus ) 的目标黄体生成素 ( lh )基因座中分别由双链 DNA (dsDNA) 和单链寡脱氧核苷酸 (ssODN) 辅助。在本研究中，使用 ssODN 策略实现了高敲入 (KI) 效率（22.38%，64/286）但较低的靶向事件，而采用 dsDNA 作为供体模板可实现有效的靶向 KI（10.80 ％，23/213）。As-Cath的目标 KI有助于建立lh敲除 (LH ^– _As-Cath ⁺ ) 鲶鱼品系，与野生型 (WT) 同胞鱼相比，该品系表现出更高的抗病性和更低的繁殖力。此外，施用人绒毛膜促性腺激素（HCG）和黄体生成素释放激素类似物（LHRHa）可以恢复转基因鱼系的繁殖。总的来说，我们用鳄鱼抗菌素转基因取代了lh基因，然后进行激素治疗，以环境无害的方式获得抗病转基因鲶鱼的完全繁殖控制。这一策略不仅有效地改善了消费者看重的性状，而且还防止了不必要的基因渗入，为水产养殖遗传学提供了突破，以限制鱼类繁殖并防止自然环境中转基因或家养基因型的建立。