High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases
High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases
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DOI：
10.1038/s41467-019-09987-0
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发表时间：
2019-04-26
影响因子：
16.6
通讯作者：
Liu, David R.
中科院分区：
文献类型：
--
作者：
Bessen, Jeffrey L.;Afeyan, Lena K.;Liu, David R.
位点特异性重组酶（SSR）作为基因组编辑剂的发展受到改变其天然DNA特异性的困难的限制。在这里，我们描述了Rec-seq，一种以全面和公正的方式揭示SSR的DNA特异性决定簇和潜在脱靶底物的方法。我们应用Rec-seq来表征几个天然和进化的SSR的DNA特异性决定簇，包括Cre，Cre的进化变体和其他SSR家族成员。这些酶及其突变体的Rec-seq分析揭示了先前未表征的SSR相互作用，包括从SSR：DNA结构中不明显的特异性决定簇。最后，我们使用Rec-seq特异性谱来预测Tre和Brec 1重组酶的脱靶底物，包括内源性人类基因组序列，并证实了它们在人类细胞中重组这些脱靶序列的能力。这些发现确立了Rec-seq作为一种高分辨率方法，用于快速表征具有单核苷酸分辨率的重组酶的DNA特异性，并为其进一步发展提供信息。
The development of site-specific recombinases (SSRs) as genome editing agents is limited by the difficulty of altering their native DNA specificities. Here we describe Rec-seq, a method for revealing the DNA specificity determinants and potential off-target substrates of SSRs in a comprehensive and unbiased manner. We applied Rec-seq to characterize the DNA specificity determinants of several natural and evolved SSRs including Cre, evolved variants of Cre, and other SSR family members. Rec-seq profiling of these enzymes and mutants thereof revealed previously uncharacterized SSR interactions, including specificity determinants not evident from SSR: DNA structures. Finally, we used Rec-seq specificity profiles to predict off-target substrates of Tre and Brec1 recombinases, including endogenous human genomic sequences, and confirmed their ability to recombine these off-target sequences in human cells. These findings establish Rec-seq as a high-resolution method for rapidly characterizing the DNA specificity of recombinases with single-nucleotide resolution, and for informing their further development.