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工程化的V-K型cast可精确剪切和粘贴DNA插入
2023-01-05 14:40

美国马萨诸塞州总医院Benjamin P. Kleinstiver研究组使用工程化的V-K型 CRISPR相关转座酶(cast)精确剪切和粘贴DNA插入。2023年1月2日出版的《自然—生物技术》杂志发表了这项成果。

他们通过设计新的铸件,提高集成产品纯度和全基因组特异性。他们设计了一个与TnsB(命名为HELIX)融合的切口归巢内切酶,以恢复DNA供体上货物切除所需的5 '切口能力。HELIX可实现DNA剪切粘贴插入,简单插入产品纯度高达99.4%,同时对基因组靶标保持稳健的集成效率。HELIX比典型的cast具有更高的靶向特异性,并且他们确定了几个新的因素,进一步调节靶向和全基因组整合。最后,他们将HELIX扩展到其他V-K型同源序列,并证明了HELIX介导的整合在人类细胞环境中的可行性。

据介绍,cast能够在RNA编程的基因组位置上进行与重组无关的多千碱基DNA插入。然而,V-K型铸件的应用受到高度脱靶整合和转位机制的阻碍,转位机制导致所需的简单货物插入和不需要的质粒协整产物的混合。

附:英文原文

Title: Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases

Author: Tou, Connor J., Orr, Benno, Kleinstiver, Benjamin P.

Issue&Volume: 2023-01-02

Abstract: CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. However, the utility of type V-K CASTs is hindered by high off-target integration and a transposition mechanism that results in a mixture of desired simple cargo insertions and undesired plasmid cointegrate products. Here we overcome both limitations by engineering new CASTs with improved integration product purity and genome-wide specificity. To do so, we engineered a nicking homing endonuclease fusion to TnsB (named HELIX) to restore the 5′ nicking capability needed for cargo excision on the DNA donor. HELIX enables cut-and-paste DNA insertion with up to 99.4% simple insertion product purity, while retaining robust integration efficiencies on genomic targets. HELIX has substantially higher on-target specificity than canonical CASTs, and we identify several novel factors that further regulate targeted and genome-wide integration. Finally, we extend HELIX to other type V-K orthologs and demonstrate the feasibility of HELIX-mediated integration in human cell contexts.

DOI: 10.1038/s41587-022-01574-x

Source: https://www.nature.com/articles/s41587-022-01574-x

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


本期文章:《自然—生物技术》:Online/在线发表

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