2026年7月22日出版的《自然》杂志发表了中国科学家的一项最新研究成果。来自清华大学的王海峰小组的最新研究提出了高效和精确的可编程DNA敲入，没有双链断裂。

在这里，研究小组报道了CRISPR千碱基级缺口酶靶向（KNIT）编辑，用于高效、精确和可编程的千碱基级DNA插入，而不需要双链DNA切割，这是通过Cas9缺口酶与DNA供体招募系统的偶联实现的。针织编辑促进DNA片段的可编程集成从0.7KB到10多个KB和有效跨越基因组位点和细胞类型。它的效率高达89%，显著降低了意外插入-删除突变（indels）率、易位和脱靶编辑。该系统支持重复插入编辑和多位点基因敲入与最小易位。它的增强版KNIT编辑器2、通过单次转染进一步提高效率。

此外，在具有病理性突变的突变细胞中，KNIT编辑通过将治疗性基因插入其安全位点或其天然位点来恢复正常的基因表达。值得注意的是，KNIT编辑使非病毒和可编程嵌合抗原受体T细胞(CAR-T没有双链断裂和临床相关效率的细胞工程。

此外，经过改造的CAR-T细胞在体外和无主题模型中表现出有效的抗肿瘤活性。因此，通过在没有双链断裂的情况下实现可编程和位点特异性千碱基规模的DNA插入，同时减少意外结果，KNIT编辑为推进个性化医疗提供了一个多功能平台。

据了解，可编程基因敲入在治疗遗传疾病和推进细胞疗法方面有着巨大的希望。然而，实现精确和高效的千碱基尺度DNA片段整合仍然具有挑战性。

附：英文原文

Title: Efficient and precise programmable DNA knock-in without double-strand breaks

Author: Gao, Yanmin, Ma, Yu, Yu, Kexin, Liu, Yintian, Gu, Buming, Tang, Hao, Yan, Wenjie, Yang, Shuangshuang, Su, Jingran, Wang, Xindong, Ma, Xin, Wang, Xinming, Wang, Fang, Li, Qingyang, Liu, Mengying, Wang, Haifeng

Issue&Volume: 2026-07-22

Abstract: Programmable gene knock-in holds substantial promise for treating genetic diseases and advancing cell therapies. However, achieving precise and efficient kilobase-scale DNA fragment integration remains challenging1,2. Here we report CRISPR kilobase-scale nickase-targeting (KNIT) editing for efficient, precise and programmable kilobase-scale DNA insertion without double-strand DNA cleavage, which is enabled through the coupling of a Cas9 nickase with a DNA donor recruiting system. KNIT editing facilitates programmable integration of DNA fragments from 0.7kb to more than 10kb and is effective across genomic loci and cell types. It achieves up to 89% efficiency and markedly reduces unintended insertion-deletion mutation (indels) rates, translocations and off-target editing. The system supports repeated insertion editing and multiloci gene knock-in with minimal translocations. Its enhanced version, KNIT editor2, further improves efficiency via a single transfection. Moreover, in mutant cells with a pathological mutation, KNIT editing restores normal gene expression by inserting a therapeutic gene into a safe harbour locus or its native locus. Notably, KNIT editing enables non-viral and programmable chimeric antigen receptor T cell (CAR-Tcell) engineering without double-strand breaks and with clinically relevant efficiencies. Moreover, the engineered CAR-Tcells exhibit effective antitumour activity in vitro and in mouse models. Therefore, by achieving programmable and site-specific kilobase-scale DNA insertions without double-strand breaks while reducing unintended outcomes, KNIT editing provides a versatile platform for advancing personalized medicine.

DOI: 10.1038/s41586-026-10819-7

Source: https://www.nature.com/articles/s41586-026-10819-7

期刊信息

Nature：《自然》，创刊于1869年。隶属于施普林格·自然出版集团，最新IF：69.504

官方网址：http://www.nature.com/

投稿链接：http://www.nature.com/authors/submit_manuscript.html