Exploiting simultaneous positive and negative selection to advance directed evolution of orthogonal RNA-guided nucleases

项目摘要 可编程的RNA引导的核酸酶正在促进自上而下的基因组编辑工作 在生物医学研究界，并显示出在治疗应用中的巨大潜力。该序列 常用的RNA引导核酸酶，包括化脓性链球菌Cas9(SpCas9)，其特异性是 由RNA-DNA碱基配对和与靶标邻接DNA序列的基本相互作用共同实现 称为ProtSpacer相邻基序(PAM)。尽管SpCas9几乎可以容纳任何引导RNA 在序列中，ITS与PAM的序列特异性相互作用总是由蛋白质-DNA接触决定。 因此，SPCas9的S的整体可编程性受到其固有的PAM需求的限制，这可能会限制 编辑需要非常精确的目标站点定位的应用程序。 以前的研究已经使用了蛋白质工程的程序，包括定向进化，以改变 RNA引导的核酸酶的蛋白质成分，如SpCas9以各种方式。虽然不是详尽的，但其中一些 这些研究已经成功地产生了具有某些替代PAM特异性的SpCas9变体。其中 到目前为止，测试的定向进化疗法涉及定制的微生物系统，这些系统被设计成将 SpCas9‘S PAM体内特异性阳性筛选正向选择足以推动 具有松弛专一性的蛋白质变体，以及具有真正改变的、正交专一性的变体。鉴于 松弛的PAM特异性扩大了可用于靶向的基因组序列空间，可能是理想的 典型的编辑应用程序，在某些等位基因特定的编辑应用程序中仍然使用正交变体 其中PAM要求可以被用来区分仅有一个 核苷酸。为了有利于正交变体的进化，定向进化过程可以结合 对保留亲本底物的变异体进行反选择的负选择压力 偏好。这些策略是否对工程设计正交PAM特性有效，目前尚不清楚 然而，已经被调查了。 这项拟议研究的目标是建立和评估微生物定向进化方案，这些方案将 同时正负选择或单独正选择对SpCas9‘S PAM特异性的影响。侧面- 通过对在以下方面发展的各种池进行高吞吐量性能分析，将实现对这些过程的附带评估 否定选择的存在或不存在，以及突变解剖和克隆标杆 化验。总的来说，这些实验将确保确定最理想的SpCas9变体，以及 指导旨在产生正交蛋白质的定向进化过程的系统改进 功能。

Project Summary Programmable RNA-guided nucleases are facilitating top-down genome editing endeavors throughout the biomedical research community, and show great potential for use in therapeutic applications. The sequence specificity of commonly used RNA-guided nucleases, including Streptococcus pyogenes Cas9 (SpCas9), is jointly enabled by RNA-DNA base pairing and essential interactions with a target-abutting DNA sequence known as a protospacer adjacent motif (PAM). Although SpCas9 can accommodate virtually any guide RNA sequence, its sequence-specific interactions with PAMs are invariably determined by protein-DNA contacts. SpCas9’s overall programmability is therefore constrained by its intrinsic PAM requirement, and this can limit editing applications that require very precise target site positioning. Previous studies have employed procedures for protein engineering, including directed evolution, to alter the protein component of RNA-guided nucleases such as SpCas9 in various ways. While not exhaustive, some of these studies have successfully produced variants of SpCas9 with certain alternative PAM specificities. Among these, the directed evolution regimens tested to date involved custom microbial systems engineered to impose positive selection on SpCas9’s PAM specificity in vivo. Positive selection is sufficient to drive the evolution of protein variants with relaxed specificity, as well as variants with truly altered, orthogonal specificity. Whereas relaxed PAM specificities that broaden the genomic sequence space accessible for targeting may be ideal for typical editing applications, orthogonal variants are still of use in certain allele-specific editing applications where the PAM requirement can be exploited to discriminate between two alleles that differ by only a single nucleotide. To favor the evolution of orthogonal variants, directed evolution procedures may incorporate negative selection pressures that counterselect against variants which retain their parental substrate preferences. Whether such strategies would be effective for engineering orthogonal PAM specificities has not been investigated, however. The goal of this proposed study is to establish and evaluate microbial directed evolution regimens that impose simultaneous positive and negative selection, or positive selection alone, on SpCas9’s PAM specificity. Side- by-side evaluation of these procedures will be enabled by high-throughput profiling of variant pools evolved in the absence or presence of negative selection, along with mutational dissections and clonal benchmarking assays. Collectively, these experiments will ensure identification of the most desirable SpCas9 variants, and guide the systematic refinement of directed evolution procedures intended to generate orthogonal protein functions.