. 2022 Apr 24;11(5):649.

doi: 10.3390/biology11050649.

Gene Editing of the Catfish Gonadotropin-Releasing Hormone Gene and Hormone Therapy to Control the Reproduction in Channel Catfish, Ictalurus punctatus

Guyu Qin¹, Zhenkui Qin^{1

2}, Cuiyu Lu¹, Zhi Ye¹, Ahmed Elaswad^{1

3}, Max Bangs^{1

4}, Hanbo Li¹, Yiliu Zhang^{1

5}, Yingqi Huang^{1

5}, Huitong Shi¹, Kamal Gosh^{1

6}, Nermeen Y Abass^{1

7}, Khoi Vo¹, Ramjie Odin^{1

8}, William S Bugg^{1

9}, Nathan J C Backenstose^{1

10}, David Drescher^{1

11}, Zachary Taylor¹, Timothy Braden¹², Baofeng Su¹, Rex A Dunham¹

Affiliations

¹ School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA.
² Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China.
³ Department of Animal Wealth Development, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt.
⁴ Department of Biological Science, Florida State University, Tallahassee, FL 32304, USA.
⁵ College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.
⁶ Department of Agriculture and Natural Resources, Langston University, Langston, OK 73050, USA.
⁷ Department of Agricultural Botany, Faculty of Agriculture Saba-Basha, Alexandria University, Alexandria 21531, Egypt.
⁸ College of Fisheries, Mindanao State University-Maguindanao, Maguindanao 9601, Philippines.
⁹ Department of Biological Sciences, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
¹⁰ Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY 14228, USA.
¹¹ Muckleshoot Indian Tribe, Keta Creek Hatchery, 34900 212th Ave SE, Auburn, WA 98092, USA.
¹² Department of Anatomy, Physiology and Pharmacology, Auburn University, Auburn, AL 36849, USA.

PMID: 35625377
PMCID: PMC9138287
DOI: 10.3390/biology11050649

Gene Editing of the Catfish Gonadotropin-Releasing Hormone Gene and Hormone Therapy to Control the Reproduction in Channel Catfish, Ictalurus punctatus

Guyu Qin et al. Biology (Basel). 2022.

. 2022 Apr 24;11(5):649.

doi: 10.3390/biology11050649.

Authors

Affiliations

¹ School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA.
² Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China.
³ Department of Animal Wealth Development, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt.
⁴ Department of Biological Science, Florida State University, Tallahassee, FL 32304, USA.
⁵ College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.
⁶ Department of Agriculture and Natural Resources, Langston University, Langston, OK 73050, USA.
⁷ Department of Agricultural Botany, Faculty of Agriculture Saba-Basha, Alexandria University, Alexandria 21531, Egypt.
⁸ College of Fisheries, Mindanao State University-Maguindanao, Maguindanao 9601, Philippines.
⁹ Department of Biological Sciences, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
¹⁰ Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY 14228, USA.
¹¹ Muckleshoot Indian Tribe, Keta Creek Hatchery, 34900 212th Ave SE, Auburn, WA 98092, USA.
¹² Department of Anatomy, Physiology and Pharmacology, Auburn University, Auburn, AL 36849, USA.

PMID: 35625377
PMCID: PMC9138287
DOI: 10.3390/biology11050649

Abstract

Transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). Targeted cfGnRH fish were sequenced and base deletion, substitution, and insertion were detected. The gene mutagenesis was achieved in 52.9% of P1 fish. P1 mutants (individuals with human-induced sequence changes at the cfGnRH locus) had lower spawning rates (20.0−50.0%) when there was no hormone therapy compared to the control pairs (66.7%) as well as having lower average egg hatch rates (2.0% versus 32.3−74.3%) except for one cfGnRH mutated female that had a 66.0% hatch rate. After low fertility was observed in 2016, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants in 2017, which were not significantly different from the controls (p > 0.05). No exogenous DNA fragments were detected in the genome of mutant P1 fish, indicating no integration of the plasmids. No obvious effects on other economically important traits were observed after the knockout of the reproductive gene in the P1 fish. Growth rates, survival, and appearance between mutant and control individuals were not different. While complete knock-out of reproductive output was not achieved, as these were mosaic P1 brood stock, gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.

Keywords: catfish gonadotropin-releasing hormone; channel catfish; hormone therapy; transcription activator-like effector nucleases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Figure 1**
Identification of edited catfish-type gonadotropin-releasing hormone (cfGnRH) gene in P₁ channel catfish (*Ictalurus punctatus*) using the Surveyor mutation detection assay. The left “−” indicates the negative control without template. The right “−” indicates the negative control with wild-type template; “m” indicates 1 kb DNA ladder; 2, 3, 4 and 5 are channel catfish with mutation; Lane 1 is a channel catfish without the mutation. Figure 1 was cropped from the full-length gels that were presented in Figure S2.

**Figure 2**
Nucleic acid sequences and corresponding predicted amino acid sequences of catfish-type gonadotropin-releasing hormone (cfGnRH) gene in wild-type channel catfish (*Ictalurus punctatus*) and after edited with transcription activator-like effector nucleases (TALENs). (A) EX1…EX4 indicate exon1…exon4. The wild-type channel catfish cfGnRH gene sequence is shown on the top. Sequences in orange are the target binding sites of the TALENs. Sequences (blue highlighted) in the middle portion of gonadotropin-releasing hormone associated peptide (GAP) are the expected cleavage sites of the nucleases. Red dashes and letters indicate the deletion, insertion/substitution of nucleotides. Numbers at the end of the sequences show the number of nucleotides deleted (−) or inserted (+) in the edited cfGnRH gene. (B) Predicted amino acid sequences with incomplete domain were due to frame-shift reading, resulting in a premature stop (red color) codon. Single amino acid substitutions or deletions (red color) were due to single nucleotide substitution mutation or three nucleotides deletion.

**Figure 3**
Polymerase chain reaction (PCR) inspection of potential transcription activator-like effector nuclease (TALEN) plasmid integration into channel catfish (*Ictalurus punctatus*) genome. In (A,B), the left lanes of “−” indicate the negative controls without template, while the right lanes of “−” indicate the negative controls with wild-type channel catfish DNA as a template; “+” indicates the positive controls with left and right TALEN plasmids as a template, respectively; “m” indicates 1 kb DNA ladders. Numbers represent samples from channel catfish individuals carrying mutated cfGnRH gene; the same number indicates the same individual in (A,B). (A,B) represent the PCR detection with different specific primers designed to amplify the cytomegalovirus (CMV) promoter region and the transcription activator-like (TAL) repeats region, respectively. (A,B) were cropped from the full-length gels that were presented in Figure S3.

**Figure 4**
Images of the spawned P₁ channel catfish (*Ictalurus punctatus*) in the aquarium, and the egg masses with high hatch rate and low hatch rate, respectively. (A) Showed the spawned P₁ fish with normal behavior and egg mass. (B) Showed the egg mass with high hatch rate. (C) Showed the egg mass with low hatch rate.

**Figure 5**
Identification of edited catfish-type gonadotropin-releasing hormone (cfGnRH) gene in F₁ channel catfish (*Ictalurus punctatus*) using the Surveyor mutation detection assay. The negative control is not shown in this image. “m” indicates 1 kb DNA ladder; 1, 2, 3, 4 and 5 are channel catfish with mutation. Figure 5 was cropped from the full-length gels that were presented in Figure S4.

See this image and copyright information in PMC

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References

1. Liu Z., Liu S., Yao J., Bao L., Zhang J., Li Y., Jiang C., Sun L., Wang R., Zhang Y., et al. The Channel Catfish Genome Sequence Provides Insights into the Evolution of Scale Formation in Teleosts. Nat. Commun. 2016;7:11757. doi: 10.1038/ncomms11757. - DOI - PMC - PubMed
1. Dong S., Perera D.A., Vo K., Odin R., Alsaquifi A., Fobes M., Elaswad A., Ye Z., Qin Z., Li H., et al. Channel Catfish Ictalurus punctatus Strain Comparison for Induced Ovulation in the Early Spawning Season to Produce Channel Catfish ♀×blue Catfish, I. Furcatus ♂ Hybrid Catfish Embryos. Aquaculture. 2017;471:185–189. doi: 10.1016/j.aquaculture.2016.11.029. - DOI
1. U.S. Catfish Database. 2014. [(accessed on 13 March 2022)]. Available online: https://www.agecon.msstate.edu/whatwedo/budgets/docs/catfish2014.pdf.
1. Abass N.Y., Elwakil H.E., Hemeida A.A., Abdelsalam N.R., Ye Z., Su B., Alsaqufi A.S., Weng C.-C., Trudeau V.L., Dunham R.A. Genotype–Environment Interactions for Survival at Low and Sub-Zero Temperatures at Varying Salinity for Channel Catfish, Hybrid Catfish and Transgenic Channel Catfish. Aquaculture. 2016;458:140–148. doi: 10.1016/j.aquaculture.2016.02.031. - DOI
1. Abdelrahman H., The Aquaculture Genomics, Genetics and Breeding Workshop. ElHady M., Alcivar-Warren A., Allen S., Al-Tobasei R., Bao L., Beck B., Blackburn H., Bosworth B., et al. Aquaculture Genomics, Genetics and Breeding in the United States: Current Status, Challenges, and Priorities for Future Research. BMC Genom. 2017;18:191. doi: 10.1186/s12864-017-3557-1. - DOI - PMC - PubMed

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Gene Editing of the Catfish Gonadotropin-Releasing Hormone Gene and Hormone Therapy to Control the Reproduction in Channel Catfish, Ictalurus punctatus

Affiliations

Gene Editing of the Catfish Gonadotropin-Releasing Hormone Gene and Hormone Therapy to Control the Reproduction in Channel Catfish, Ictalurus punctatus

Authors

Affiliations

Abstract

Conflict of interest statement

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