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Generation of Eco-Friendly and Disease-Resistant Channel Catfish(Ictalurus punctatus)Harboring the Alligator Cathelicidin Gene via CRISPR/Cas9 Engineering

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Engineering 2024年第8期39卷 273-286页

作者： Jinhai Wang Baofeng Su De Xing Timothy J.Bruce Shangjia Li Logan Bern Mei Shang Andrew Johnson Rhoda Mae C.Simora Michael Coogan darshika u.hettiarachchi Wenwen Wang Tasnuba Hasin Jacob Al-Armanazi Cuiyu Lu Rex A.Dunham School of Fisheries Aquacultureand Aquatic SciencesAuburn UniversityAuburnAL 36849USA

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 *** 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 *** 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),*** this study,high knock in(KI)efficiency(22.38%,64/286)but low ontarget 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 ***,administration of human chorionic gonadotropin(HCG)and luteinizing hormone-releasing hormone analogue(LHRHa)can restore the reproduction of the transgenic fish ***,we replaced the lh gene with an alligator cathelicidin transgene and then administered hormone therapy to move towards complete reproductive control of diseaseresistant transgenic catfish in an environmentally responsible *** 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.

关键词： Genome editing ssODN dsDNA Antimicrobial peptide Reproductive confinement Aquaculture

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