Juillerat Alexandre, Pessereau Coline, Dubois Gwendoline, Guyot Valérie, Maréchal Alan, Valton Julien, Daboussi Fayza, Poirot Laurent, Duclert Aymeric, Duchateau Philippe
CELLECTIS S.A., 8 Rue de la Croix Jarry, 75013 Paris, France.
Sci Rep. 2015 Jan 30;5:8150. doi: 10.1038/srep08150.
A key feature when designing DNA targeting tools and especially nucleases is specificity. The ability to control and tune this important parameter represents an invaluable advance to the development of such molecular scissors. Here, we identified and characterized new non-conventional RVDs (ncRVDs) that possess novel intrinsic targeting specificity features. We further report a strategy to control TALEN targeting based on the exclusion capacities of ncRVDs (discrimination between different nucleotides). By implementing such ncRVDs, we demonstrated in living cells the possibility to efficiently promote TALEN-mediated processing of a target in the HBB locus and alleviate undesired off-site cleavage. We anticipate that this method can greatly benefit to designer nucleases, especially for therapeutic applications and synthetic biology.
设计DNA靶向工具尤其是核酸酶时的一个关键特性是特异性。控制和调节这一重要参数的能力是此类分子剪刀发展的一项极有价值的进展。在此，我们鉴定并表征了具有新型内在靶向特异性特征的新型非常规RVD（ncRVD）。我们进一步报告了一种基于ncRVD的排斥能力（区分不同核苷酸）来控制TALEN靶向的策略。通过应用此类ncRVD，我们在活细胞中证明了有效促进TALEN介导的HBB基因座中靶标的加工以及减轻不期望的脱靶切割的可能性。我们预计这种方法可极大地有益于设计核酸酶，特别是在治疗应用和合成生物学方面。
