利用工程重组酶将位点特异性DNA插入人类基因组，这一成果由美国加州大学Patrick D. Hsu研究组经过不懈努力而取得。这一研究成果发表在2025年11月6日出版的国际学术期刊《自然—生物技术》上。

在这里，该团队提出了一个共同优化DNA重组效率和特异性的工程路线图。研究团队结合了定向进化，结构分析和计算模型来快速识别加性突变组合。研究小组通过供体DNA优化和基因编辑进一步提高了它们的性能，使它们能够同时招募靶标和供体。他们的顶级LSR变种，superDn29dCas9、goldDn29 dCas9和hifin29 dCas9，在内源性人类位点上实现高达53%的整合效率和97%的全基因组特异性，并有效整合多达12kb在非分裂细胞、干细胞和原代人T细胞中稳定表达。DNA重组酶的合理工程设计使精确和高效的单步基因组插入成为基因和细胞治疗的多种应用。

据了解，将大的DNA序列插入到基因组中广泛地用于研究和治疗应用。大丝氨酸重组酶（Large serine recombinases, lsr）可以介导多千碱基DNA序列的直接、位点特异性基因组整合，而无需预先安装着陆垫，尽管其插入率低，脱靶活性高。

附：英文原文

Title: Site-specific DNA insertion into the human genome with engineered recombinases

Author: Fanton, Alison, Bartie, Liam J., Martins, Juliana Q., Tran, Vincent Q., Goudy, Laine, Kernick, Courtney, Durrant, Matthew G., Wei, Jingyi, Armour-Garb, Zev, Pawluk, April, Konermann, Silvana, Marson, Alexander, Gilbert, Luke A., Roth, Theodore L., Hsu, Patrick D.

Issue&Volume: 2025-11-06

Abstract: Insertions of large DNA sequences into the genome are broadly enabling for research and therapeutic applications. Large serine recombinases (LSRs) can mediate direct, site-specific genomic integration of multi-kilobase DNA sequences without a pre-installed landing pad, albeit with low insertion rates and high off-target activity. Here we present an engineering roadmap for jointly optimizing their DNA recombination efficiency and specificity. We combine directed evolution, structural analysis and computational models to rapidly identify additive mutational combinations. We further enhance performance through donor DNA optimization and dCas9 fusions, enabling simultaneous target and donor recruitment. Our top engineered LSR variants, superDn29dCas9, goldDn29dCas9 and hifiDn29dCas9, achieve up to 53% integration efficiency and 97% genome-wide specificity at an endogenous human locus and effectively integrate large DNA cargoes up to 12kb for stable expression in non-dividing cells, stem cells and primary human T cells. Rational engineering of DNA recombinases enables precise and efficient single-step genome insertion for diverse applications across gene and cell therapies.

DOI: 10.1038/s41587-025-02895-3

Source: https://www.nature.com/articles/s41587-025-02895-3