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

利用同时正向和负向选择来推进正交 RNA 引导核酸酶的定向进化

• 批准号: 10133453
• 负责人: Gregory William Goldberg
• 金额: $ 6.86万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10133453
• 关键词: Alleles; Amino Acid Sequence; Bacteria; Base Pairing; Benchmarking; Biological; Biological Assay; Biomedical Research; Bypass; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Coupled; Coupling; Custom; DNA; DNA Sequence; Development; Directed Molecular Evolution; Disadvantaged; Dissection; Engineering; Ensure; Escherichia coli; Evaluation; Evolution; Exhibits; Future; General Practitioners; Genome; Genomics; Goals; Guide RNA; Guidelines; Harvest; High-Throughput Nucleotide Sequencing; Laboratories; Libraries; Microbe; Modeling; Mutagenesis; Mutation; Nucleotides; Parents; Positioning Attribute; Procedures; Protein Engineering; Proteins; RNA; RNA Sequences; Reagent; Regimen; Reporting; Saccharomyces cerevisiae; Selection Criteria; Side; Site; Specificity; Streptococcus pyogenes; System; Testing; Therapeutic; Variant; Work; Yeasts; base; comparative; design; experimental study; genome editing; in vivo; insight; interest; microbial; mutant; novel; nuclease; preference; pressure; programs; protein function; screening; synthetic protein; virtual

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.

项目摘要 可编程的RNA引导的核酸酶促进了整个基因组中自上而下的基因组编辑努力。 生物医学研究社区，并显示出巨大的潜力，用于治疗应用。序列 通常使用的RNA引导的核酸酶，包括化脓性链球菌Cas9（SpCas 9）的特异性， 通过RNA-DNA碱基配对和与靶邻接DNA序列的必要相互作用共同实现 称为原型间隔区邻近基序（PAM）。尽管SpCas 9可以容纳几乎任何向导RNA， 序列，其与PAM的序列特异性相互作用总是由蛋白质-DNA接触决定。 因此，SpCas 9的整体可编程性受到其固有PAM要求的约束，这可能限制了SpCas 9的可编程性。 编辑需要非常精确的目标位置定位的应用程序。 以前的研究已经采用蛋白质工程的程序，包括定向进化，以改变蛋白质的结构。 RNA引导的核酸酶如SpCas 9的蛋白质组分。虽然不是详尽无遗的， 这些研究已经成功地产生了具有某些替代PAM特异性的SpCas 9变体。之间 这些，迄今为止测试的定向进化方案涉及定制的微生物系统， 在体内对SpCas 9的PAM特异性进行阳性选择。积极的选择足以推动进化， 具有松弛特异性的蛋白变体，以及具有真正改变的正交特异性的变体。而 放宽的PAM特异性拓宽了可用于靶向的基因组序列空间， 尽管在典型的编辑应用中，正交变体仍用于某些等位基因特异性编辑应用中 其中PAM要求可用于区分仅相差单个等位基因的两个等位基因， 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 负选择压力，反选择保留其亲本底物的变体 喜好这种策略是否对工程化正交PAM特异性有效还没有 然而，被调查。 这项研究的目的是建立和评价微生物定向进化方案， 同时的阳性和阴性选择，或单独的阳性选择对SpCas 9的PAM特异性的影响。侧- 这些程序的并行评估将通过对变异库的高通量分析来实现， 阴性选择的存在或不存在，沿着突变解剖和克隆基准 测定。总的来说，这些实验将确保鉴定最理想的SpCas 9变体，并且 指导定向进化程序的系统完善，旨在产生正交蛋白质 功能协调发展的