Comparison of Multiple Strategies for Precision Transgene Knock-In in <i>Gallus gallus</i> Genome via Microhomology-Mediated End Joining
Precision exogenous gene knock-in is an attractive field for transgenic <i>Gallus gallus</i> (chicken) generation. In this article, we constructed multiple Precise Integration into Target Chromosome (PITCh) plasmid systems mediated by microhomology-mediated end-joining (MMEJ) for large-f...
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MDPI AG
2023-10-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/21/15731 |
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| author | Lijuan Wang Jiaxin Sun Zhipeng Liu Qiang Zheng Guojun Wang |
| author_facet | Lijuan Wang Jiaxin Sun Zhipeng Liu Qiang Zheng Guojun Wang |
| author_sort | Lijuan Wang |
| collection | DOAJ |
| description | Precision exogenous gene knock-in is an attractive field for transgenic <i>Gallus gallus</i> (chicken) generation. In this article, we constructed multiple Precise Integration into Target Chromosome (PITCh) plasmid systems mediated by microhomology-mediated end-joining (MMEJ) for large-fragment integration in DF-1 cells and further assess the possibility of <i>GAPDH</i> (glyceraldehyde-3-phosphate dehydrogenase) as a genomic safe harbor for chickens. We designed three targeted sgRNAs for the all-in-one plasmid at the 3′UTR of <i>GAPDH</i> near the stop codon. The donor-plasmid-carrying microhomology arms correspond to sgRNA and EGFP fragments in the forward and reverse directions. MMEJ-mediated EGFP insertion can be efficiently expressed in DF-1 cells. Moreover, the differences between the forward and reverse fragments indicated that promoter interference does affect the transfection efficiency of plasmids and cell proliferation. The comparison of the 20 bp and 40 bp microhomology arms declared that the short one has higher knock-in efficiency. Even though all three different transgene insertion sites in <i>GAPDH</i> could be used to integrate the foreign gene, we noticed that the G2-20R-EGFP cell reduced the expression of <i>GAPDH</i>, and the G3-20R-EGFP cell exhibited significant growth retardation. Taken together, G1, located at the 3′UTR of <i>GAPDH</i> on the outer side of the last base of the terminator, can be a candidate genomic safe harbor (GSH) loci for the chicken genome. In addition, deleted-in-azoospermia-like (<i>DAZL</i>) and actin beta (<i>ACTB</i>) site-specific gene knock-in indicated that MMEJ has broad applicability and high-precision knock-in efficiency for genetically engineered chickens. |
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| language | English |
| last_indexed | 2024-03-11T11:28:34Z |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-3ce2d6e1d55245b3afd9865877b918872023-11-10T15:05:05ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-10-0124211573110.3390/ijms242115731Comparison of Multiple Strategies for Precision Transgene Knock-In in <i>Gallus gallus</i> Genome via Microhomology-Mediated End JoiningLijuan Wang0Jiaxin Sun1Zhipeng Liu2Qiang Zheng3Guojun Wang4The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot 010070, ChinaThe State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot 010070, ChinaThe State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot 010070, ChinaThe State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot 010070, ChinaThe State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot 010070, ChinaPrecision exogenous gene knock-in is an attractive field for transgenic <i>Gallus gallus</i> (chicken) generation. In this article, we constructed multiple Precise Integration into Target Chromosome (PITCh) plasmid systems mediated by microhomology-mediated end-joining (MMEJ) for large-fragment integration in DF-1 cells and further assess the possibility of <i>GAPDH</i> (glyceraldehyde-3-phosphate dehydrogenase) as a genomic safe harbor for chickens. We designed three targeted sgRNAs for the all-in-one plasmid at the 3′UTR of <i>GAPDH</i> near the stop codon. The donor-plasmid-carrying microhomology arms correspond to sgRNA and EGFP fragments in the forward and reverse directions. MMEJ-mediated EGFP insertion can be efficiently expressed in DF-1 cells. Moreover, the differences between the forward and reverse fragments indicated that promoter interference does affect the transfection efficiency of plasmids and cell proliferation. The comparison of the 20 bp and 40 bp microhomology arms declared that the short one has higher knock-in efficiency. Even though all three different transgene insertion sites in <i>GAPDH</i> could be used to integrate the foreign gene, we noticed that the G2-20R-EGFP cell reduced the expression of <i>GAPDH</i>, and the G3-20R-EGFP cell exhibited significant growth retardation. Taken together, G1, located at the 3′UTR of <i>GAPDH</i> on the outer side of the last base of the terminator, can be a candidate genomic safe harbor (GSH) loci for the chicken genome. In addition, deleted-in-azoospermia-like (<i>DAZL</i>) and actin beta (<i>ACTB</i>) site-specific gene knock-in indicated that MMEJ has broad applicability and high-precision knock-in efficiency for genetically engineered chickens.https://www.mdpi.com/1422-0067/24/21/15731MMEJ<i>GAPDH</i>DF-1 cell lineprecise integrationCRISPR/Cas9safe harbor loci |
| spellingShingle | Lijuan Wang Jiaxin Sun Zhipeng Liu Qiang Zheng Guojun Wang Comparison of Multiple Strategies for Precision Transgene Knock-In in <i>Gallus gallus</i> Genome via Microhomology-Mediated End Joining International Journal of Molecular Sciences MMEJ <i>GAPDH</i> DF-1 cell line precise integration CRISPR/Cas9 safe harbor loci |
| title | Comparison of Multiple Strategies for Precision Transgene Knock-In in <i>Gallus gallus</i> Genome via Microhomology-Mediated End Joining |
| title_full | Comparison of Multiple Strategies for Precision Transgene Knock-In in <i>Gallus gallus</i> Genome via Microhomology-Mediated End Joining |
| title_fullStr | Comparison of Multiple Strategies for Precision Transgene Knock-In in <i>Gallus gallus</i> Genome via Microhomology-Mediated End Joining |
| title_full_unstemmed | Comparison of Multiple Strategies for Precision Transgene Knock-In in <i>Gallus gallus</i> Genome via Microhomology-Mediated End Joining |
| title_short | Comparison of Multiple Strategies for Precision Transgene Knock-In in <i>Gallus gallus</i> Genome via Microhomology-Mediated End Joining |
| title_sort | comparison of multiple strategies for precision transgene knock in in i gallus gallus i genome via microhomology mediated end joining |
| topic | MMEJ <i>GAPDH</i> DF-1 cell line precise integration CRISPR/Cas9 safe harbor loci |
| url | https://www.mdpi.com/1422-0067/24/21/15731 |
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