将特定位点的反转录转座子活性重编程到新的DNA位点，这一成果由麻省理工学院Jonathan S. Gootenberg研究组经过不懈努力而取得。相关论文发表在2025年4月9日出版的《自然》杂志上。

在这里，该课题组设计了一个计算管道来发现多个新的位点特异性反转录转座子家族，在生物化学和哺乳动物细胞中分析成员，发现以前未描述的插入偏好，并绘制反转录转座子重靶向的潜在进化路径。课题组人员发现来自斑胸草雀（Taeniopygia guttata）的R2反转录转座子R2Tg是一个同源物，可以通过有效载荷工程在新的基因组位点上进行靶切割、反转录和无疤插入异源有效载荷来重新定位。

课题组研究人员通过将R2Tg修饰为CRISPR-Cas9缺口酶来增强这种活性，从而有效地插入新的基因组位点。通过进一步筛选R2同源物，该研究团队选择了一个具有天然可重编程性和在其天然28S位点插入最少的同源物R2Tocc来设计SpCas9^H840A-R2Tocc，一个该研究团队通过靶向CRISPR归巢逆转录元件（STITCHR）命名为位点特异性靶向引物插入的系统。STITCHR可以实现无疤痕，高效的编辑安装，范围从单个碱基到12.7千碱基，基因替换和体外转录或合成RNA模板的主题。受nLTR反转录转座子在真核生物基因组中普遍存在的启发，该课题组研究人员预计STITCHR将作为分裂细胞和非分裂细胞无疤痕可编程整合的平台，在研究和治疗方面都有应用。

据了解，逆转录因子在形成真核生物基因组中起着关键作用。例如，位点特异性非长末端重复反转录转座子通过优先整合到重复基因组序列中，如微卫星区和核糖体DNA基因，广泛传播。尽管这些系统广泛存在，但它们的目标限制仍不清楚。

附：英文原文

Title: Reprogramming site-specific retrotransposon activity to new DNA sites

Author: Fell, Christopher W., Villiger, Lukas, Lim, Justin, Hiraizumi, Masahiro, Tagliaferri, Dario, Yarnall, Matthew T. N., Lee, Anderson, Jiang, Kaiyi, Kayabolen, Alisan, Krajeski, Rohan N., Schmitt-Ulms, Cian, Ramani, Harsh, Yousef, Sarah M., Roberts, Nathaniel, Vakulskas, Christopher A., Nishimasu, Hiroshi, Abudayyeh, Omar O., Gootenberg, Jonathan S.

Issue&Volume: 2025-04-09

Abstract: Retroelements have a critical role in shaping eukaryotic genomes. For instance, site-specific non-long terminal repeat retrotransposons have spread widely through preferential integration into repetitive genomic sequences, such as microsatellite regions and ribosomal DNA genes1,2,3,4,5,6. Despite the widespread occurrence of these systems, their targeting constraints remain unclear. Here we use a computational pipeline to discover multiple new site-specific retrotransposon families, profile members both biochemically and in mammalian cells, find previously undescribed insertion preferences and chart potential evolutionary paths for retrotransposon retargeting. We identify R2Tg, an R2 retrotransposon from the zebra finch, Taeniopygia guttata, as an orthologue that can be retargeted by payload engineering for target cleavage, reverse transcription and scarless insertion of heterologous payloads at new genomic sites. We enhance this activity by fusing R2Tg to CRISPR–Cas9 nickases for efficient insertion at new genomic sites. Through further screening of R2 orthologues, we select an orthologue, R2Tocc, with natural reprogrammability and minimal insertion at its natural 28S site, to engineer SpCas9H840A–R2Tocc, a system we name site-specific target-primed insertion through targeted CRISPR homing of retroelements (STITCHR). STITCHR enables the scarless, efficient installation of edits, ranging from a single base to 12.7kilobases, gene replacement and use of in vitro transcribed or synthetic RNA templates. Inspired by the prevalence of nLTR retrotransposons across eukaryotic genomes, we anticipate that STITCHR will serve as a platform for scarless programmable integration in dividing and non-dividing cells, with both research and therapeutic applications.

DOI: 10.1038/s41586-025-08877-4

Source: https://www.nature.com/articles/s41586-025-08877-4