Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.
Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.
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DOI：
10.1038/s41587-022-01527-4
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发表时间：
2023-04
影响因子：
46.9
通讯作者：
Gootenberg, Jonathan S.
中科院分区：
文献类型：
--
作者：
Yarnall, Matthew T. N.;Ioannidi, Eleonora I.;Schmitt-Ulms, Cian;Krajeski, Rohan N.;Lim, Justin;Villiger, Lukas;Zhou, Wenyuan;Jiang, Kaiyi;Garushyants, Sofya K.;Roberts, Nathaniel;Zhang, Liyang;Vakulskas, Christopher A.;Walker, John A. I. I. I. I.;Kadina, Anastasia P.;Zepeda, Adrianna E.;Holden, Kevin;Ma, Hong;Xie, Jun;Gao, Guangping;Foquet, Lander;Bial, Greg;Donnelly, Sara K.;Miyata, Yoshinari;Radiloff, Daniel R.;Henderson, Jordana M.;Ujita, Andrew;Abudayyeh, Omar O.;Gootenberg, Jonathan S.
在没有暴露的DNA双链断裂（DSB）的DNA修复的情况下，大的、多样的DNA货物的可编程基因组整合仍然是基因组编辑中未解决的挑战。我们提出了通过位点特异性靶向元件（PASTE）的可编程添加，其使用与逆转录酶和丝氨酸整合酶融合的CRISPR-Cas9切口酶，用于靶向基因组募集和所需有效载荷的整合。我们证明了在三种人细胞系、原代T细胞和非分裂原代人肝细胞的多个基因组位点上整合了大至约36 kb的序列。为了增强PASTE，我们从宏基因组中发现了25，614个丝氨酸整合酶和同源附着位点，并设计了具有更高活性和更短识别序列的直系同源物，以实现有效的可编程整合。PASTE具有类似于或超过同源定向修复和基于非同源末端连接的方法的编辑效率，在非分裂细胞中具有活性，并且在体内具有较少的可检测的脱靶事件。PASTE扩展了基因组编辑的能力，允许在不依赖DNA修复途径的情况下插入大型多路基因。大序列被位点特异性地整合到人类基因组中，而不需要双链DNA切割。
Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks (DSBs) remains an unsolved challenge in genome editing. We present Programmable Addition via Site-specific Targeting Elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kb at multiple genomic loci across three human cell lines, primary T cells, and non-dividing primary human hepatocytes. To augment PASTE, we discover 25,614 serine integrases and cognate attachment sites from metagenomes and engineer orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology directed repair and non-homologous end joining–based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways. Large sequences are integrated site-specifically into the human genome without double-strand DNA cleavage.
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影响因子：
46.9
作者：
Azuma, Hisaya;Paulk, Nicole;Grompe, Markus
通讯作者：
Grompe, Markus
影响因子：
12.3
作者：
Dang Y;Jia G;Choi J;Ma H;Anaya E;Ye C;Shankar P;Wu H
通讯作者：
Wu H
影响因子：
64.8
作者：
Eyquem J;Mansilla-Soto J;Giavridis T;van der Stegen SJ;Hamieh M;Cunanan KM;Odak A;Gönen M;Sadelain M
通讯作者：
Sadelain M
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
DOI：
10.1073/pnas.1533177100
发表时间：
2003-07-22
影响因子：
11.1
作者：
Akopian, A;He, JY;Stark, WM
通讯作者：
Stark, WM