Wolfs Jason M, Hamilton Thomas A, Lant Jeremy T, Laforet Marcon, Zhang Jenny, Salemi Louisa M, Gloor Gregory B, Schild-Poulter Caroline, Edgell David R
Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
Robarts Research Institute, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5B7, Canada.
Proc Natl Acad Sci U S A. 2016 Dec 27;113(52):14988-14993. doi: 10.1073/pnas.1616343114. Epub 2016 Dec 12.
The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.
CRISPR/Cas9核酸酶通常用于制造基因敲除。切割产生的平端DNA末端可通过经典的非同源末端连接修复途径(c-NHEJ)有效连接,从而使靶位点再生。这种修复产生了一个切割、连接和靶位点再生的循环,该循环会持续下去,直到通过替代NHEJ对DNA断裂进行足够的修饰以阻止Cas9进一步切割,从而产生基因敲除典型的插入和缺失异质群体。在此,我们开发了一种策略来摆脱这个循环,并通过创建一种RNA引导的双活性位点核酸酶,使其在靶位点产生两个不兼容的DNA断裂,有效地删除大部分靶位点,使其无法再生,从而使事件偏向于特定长度的缺失。TevCas9核酸酶是I-TevI核酸酶结构域与Cas9的融合体,在HEK293细胞中功能强大,以高达40%的频率产生33至36bp的缺失。深度测序显示TevCas9产物的加工极少,这与c-NHEJ途径对DNA末端的外切核酸酶降解和修复的保护作用一致。定向进化实验确定了具有更广泛靶向范围的I-TevI变体,使TevCas9成为一种易于使用的试剂。我们的结果突出了如何利用I-TevI归巢内切核酸酶的序列耐受切割特性来增强Cas9的应用,规避切割和连接循环,并使基因组编辑事件偏向于特定长度的缺失。