小柯机器人

美国加州大学Patrick D. Hsu和斯坦福大学Ami S. Bhatt等研究人员共同合作近期取得重要工作进展，他们系统的搜寻了能将大的DNA序列有效整合到人类基因组中的重组酶。该项研究成果2022年10月10日在线发表于《自然—生物技术》杂志上。

研究人员开发了一种计算方法来识别LSRs及其DNA附着位点的需求，将已知的LSRs多样性扩大了100倍，并能够预测其插入位点的特异性。研究人员测试了它们在人类细胞中的重组活性，将它们分类为“着陆垫”型、基因组靶向型或多靶标型LSRs。他们实现了比Bxb1高7倍的重组活性，对于超过7.5 kb的片段也能达到40-75%的基因组整合效率。

研究人员还展示了无病毒，直接整合质粒或扩增子库以改进功能基因组学的应用。这一直接从微生物测序数据中系统发现的重组酶提供了超过60个在人类细胞中实验表征的LSRs，在不产生DNA双链断裂的情况下用于大片段基因组插入。

据介绍，大丝氨酸重组酶（LSRs）是一种DNA整合酶，可促进移动遗传元件在细菌基因组中的位点特异性整合。迄今为止，只有少数LSRs，如Bxb1和PhiC31被鉴定报导，并且作为人类细胞DNA整合工具的效率有限。

附：英文原文

Title: Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome

Author: Durrant, Matthew G., Fanton, Alison, Tycko, Josh, Hinks, Michaela, Chandrasekaran, Sita S., Perry, Nicholas T., Schaepe, Julia, Du, Peter P., Lotfy, Peter, Bassik, Michael C., Bintu, Lacramioara, Bhatt, Ami S., Hsu, Patrick D.

Issue&Volume: 2022-10-10

Abstract: Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40–75% with cargo sizes over 7kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.

DOI: 10.1038/s41587-022-01494-w

Source: https://www.nature.com/articles/s41587-022-01494-w

Nature Biotechnology：《自然—生物技术》，创刊于1996年。隶属于施普林格·自然出版集团，最新IF：68.164
官方网址：https://www.nature.com/nbt/
投稿链接：https://mts-nbt.nature.com/cgi-bin/main.plex