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Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing
Nature Communications ( IF 16.6 ) Pub Date : 2022-06-23 , DOI: 10.1038/s41467-022-31386-1
Duško Lainšček 1, 2 , Vida Forstnerič 1 , Veronika Mikolič 3, 4 , Špela Malenšek 1, 4 , Peter Pečan 1, 4 , Mojca Benčina 1, 2 , Matjaž Sever 3, 5 , Helena Podgornik 3, 6 , Roman Jerala 1, 2
Affiliation  

The CRISPR/Cas system has emerged as a powerful and versatile genome engineering tool, revolutionizing biological and biomedical sciences, where an improvement of efficiency could have a strong impact. Here we present a strategy to enhance gene editing based on the concerted action of Cas9 and an exonuclease. Non-covalent recruitment of exonuclease to Cas9/gRNA complex via genetically encoded coiled-coil based domains, termed CCExo, recruited the exonuclease to the cleavage site and robustly increased gene knock-out due to progressive DNA strand recession at the cleavage site, causing decreased re-ligation of the nonedited DNA. CCExo exhibited increased deletion size and enhanced gene inactivation efficiency in the context of several DNA targets, gRNA selection, Cas variants, tested cell lines and type of delivery. Targeting a sequence-specific oncogenic chromosomal translocation using CCExo in cells of chronic myelogenous leukemia patients and in an animal model led to the reduction or elimination of cancer, establishing it as a highly specific tool for treating CML and potentially other appropriate diseases with genetic etiology.



中文翻译:

基于卷曲螺旋异二聚体的外切核酸酶募集到 CRISPR/Cas 以增强基因编辑

CRISPR/Cas 系统已成为一种功能强大且用途广泛的基因组工程工具,彻底改变了生物和生物医学科学,效率的提高可能会产生重大影响。在这里,我们提出了一种基于 Cas9 和核酸外切酶的协同作用来增强基因编辑的策略。通过基因编码的基于卷曲螺旋的结构域(称为 CCExo)将核酸外切酶非共价募集到 Cas9/gRNA 复合物,将核酸外切酶募集到切割位点,并且由于切割位点进行性 DNA 链衰退导致基因敲除显着增加,导致减少重新连接未编辑的 DNA。CCExo 在多个 DNA 靶标、gRNA 选择、Cas 变体、测试细胞系和递送类型的背景下表现出增加的缺失大小和增强的基因失活效率。

更新日期:2022-06-24
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