Λεπτομέρειες βιβλιογραφικής εγγραφής
| Τίτλος: |
Cas12a介导的猪细胞定点插入体系构建与效率评估. (Chinese) |
| Alternate Title: |
Construction and Efficiency Evaluation of the Cas12a-mediated Sitespecific Knock-in System in Pig Cells. (English) |
| Συγγραφείς: |
曾亚丹, 毕佳俊, 李兴龙, 常悦, 角德灵, 魏红江, 赵恒 |
| Πηγή: |
Biotechnology Bulletin; Aug2026, Vol. 42 Issue 8, p72-80, 9p |
| Θεματικοί όροι: |
Genome editing, DNA repair, Animal cell biotechnology, CRISPRs |
| Abstract (English): |
【Objective】 This study aimed to systematically evaluate the potential of the CRISPR-Cas12a (Cpf1) system mediating sitespecific gene knock-in in porcine cells, thereby providing an experimental basis and theoretical reference for its application in precise porcine genome engineering.【Method】Expression vectors for Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a) were constructed. Specific crRNAs were designed to target the well-characterized porcine "safe harbor" loci, Rosa26 and AAVS1, to compare the cleavage efficiencies of the two Cas12a variants.【Result】At the AAVS1 locus, the editing efficiencies of two crRNAs for AsCas12a were 2.6% and 1.2%, respectively, whereas those for LbCas12a were 6.5% and 2.5%. At the Rosa26 locus, the editing efficiencies for AsCas12a and LbCas12a were 5.0% and 12.7%, respectively, suggesting that LbCas12a possessed relatively higher editing activity in porcine cells. Based on these findings, knock-in donors were developed to utilize four distinct DNA repair pathways: Homology-directed repair (HDR), homology-mediated end joining (HMEJ), microhomology-mediated end joining (MMEJ), and non-homologous end joining (NHEJ), and their integration efficiencies were evaluated. The results revealed that 4 repair pathways all mediated the target genes to have site-targeted integration at both Rosa26 and AAVS1 loci. Specifically, the efficiency by HDR achieved 35.0%, 25.0% by HMEJ, and 15.0% by both NHEJ and MMEJ.【Conclusion】Four site-specific integration strategies are successfully developed in porcine cells using the Cas12a gene-editing system, followed by a systematic evaluation of their efficiencies. These findings provide essential experimental evidence and references for expanding the application of the Cas12a system in porcine genetic engineering. [ABSTRACT FROM AUTHOR] |
| Abstract (Chinese): |
【目的】系统评估 CRISPR-Cas12a (Cpf1) 系统在猪细胞中介导基因定点插入 (knock-in) 的潜能, 并为其在猪基因组 精准编辑中的应用提供实验依据与理论支撑。【方法】构建基于 Acidaminococcus sp. Cas12a (AsCas12a) 与 Lachnospiraceae bacterium Cas12a (LbCas12a) 的表达载体, 并针对猪基因组安全位点 Rosa26 与 AAVS1 设计特异性 crRNA, 旨在比较两种 Cas12a 变体的切割 效能。【结果】在 AAVS1 位点, AsCas12a 的两条 crRNA 编辑效率分别为 2.6% 与 1.2%, 而 LbCas12a 对应的效率分别为 6.5% 与 2.5% ; 在 Rosa26 位点, AsCas12a 与 LbCas12a 的编辑效率分别为 5.0% 与 12.7%, LbCas12a 在猪源细胞中展现出相对较高的编辑活性。在此 基础上, 构建了基于同源重组修复 (HDR) 、同源介导末端连接 (HMEJ) 、微同源介导末端连接 (MMEJ) 及非同源末端连接 (NHEJ) 4 种策略的敲入载体, 并评估了其整合效率。结果表明, 上述 4 种修复途径均可介导目标基因在 Rosa26 和 AAVS1 位点的定 点整合, 其中 HDR 效率为 35.0% 、HMEJ 为 25.0% 、NHEJ 与 MMEJ 均为 15.0%。【结论】基于 Cas12a 基因编辑系统, 在猪细胞中成 功构建了 4 种定点插入策略, 并对其效率进行了系统评估, 该研究成果为拓展 Cas12a 系统在猪基因工程领域的应用提供了重要的 实验依据和参考资料。 [ABSTRACT FROM AUTHOR] |
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| Βάση Δεδομένων: |
Complementary Index |