Elucidating Mechanisms of Adaptive Immunity in Type II-A CRISPR-Cas

阐明 II-A 型 CRISPR-Cas 的适应性免疫机制

• 批准号: 10214577
• 负责人: Claire T. Kenney
• 金额: $ 5.1万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10214577
• 关键词: Adaptive Immune System; Affect; BCAR1 gene; Bacteriophages; Base Pairing; Binding; Biochemical; Biological Assay; Cells; Characteristics; Cleaved cell; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; DNA; DNA Binding; DNA Sequence; Databases; Development; Devices; Elements; Eukaryota; Event; Frequencies; Genes; Genome; Heritability; Immune; Immune system; Immunity; Immunization; Immunologic Memory; In Vitro; Infection; Infection prevention; Invaded; Knowledge; Length; Libraries; Mediating; Medical; Memory; Molecular; Mutagenesis; Mutation; Nucleotides; Oligonucleotides; Patients; Plasmids; Process; Prokaryotic Cells; Proteins; Research; Role; Scanning; Specificity; Streptococcus pyogenes; System; Testing; Transcript; Viral; Virus; Work; adaptive immunity; antimicrobial; base; cancer cell; clinical application; ds-DNA; experimental study; gene function; genome editing; improved; in vitro Assay; in vivo; insight; molecular sequence database; mutant; next generation sequencing; novel; pathogen; prevent; response; treatment strategy

Project Summary CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes) functions as an adaptive immune system in prokaryotes by providing protection against bacterial viruses (phages). CRISPR loci consist of repeat sequences interspersed with short fragments of phage DNA (spacers). The CRISPR-associated (cas) genes code for proteins that regulate 1) the acquisition of new spacers during phage infection (“immunization”) and 2) the use of spacer transcripts to identify and degrade complementary foreign DNA sequences (protospacers) to prevent infection (“immunity”). My proposed research seeks to better understand the functions of the regulatory components of the Streptococcus pyogenes type II-A CRISPR-Cas system (SpyCas9) which is near ubiquitous in gene editing applications. The first aim concerns the protospacer adjacent motifs (PAMs) found in phage DNA. The canonical SpyCas9 PAM sequence is “NGG”, but non-canonical (non-NGG) PAM sequences are also seen. I collected non-NGG PAMs from spacer acquisition databases and identified those that efficiently mediated immunity. All such PAMs had the sequence “NAGG”. Thus, I hypothesize that NAGG PAMs can interact with Cas9 to mediate significant immunity. To test this, I will identify the importance of each nucleotide for immunity, as well as the immune potential of NAGGs not seen in the databases. I will also compare AGG and NAGG PAMs via direct competition during phage targeting and via libraries of all possible NAGG and AGG PAMs. Finally, I will biochemically characterize the roles of NAGG PAMs in vitro with DNA binding and cleavage assays. The second aim centers on the enigmatic SpyCas9 protein, Csn2. The other SpyCas9 Cas proteins are well- characterized, but Csn2 is known only to be essential for spacer acquisition in vivo. I investigated Csn2’s impact on the sub-steps of in vivo spacer acquisition from a cleaved protospacer prior to spacer integration into the CRISPR array. The absence of Csn2 in those stages causes a 100-fold decrease in acquisition. Hence, I hypothesize that Csn2 facilitates the selection spacers by interacting with the cleaved protospacer before it is integrated into the CRISPR array. I will test spacer acquisition from WT and Δcsn2 strains, evaluating new spacers for length, orientation, acquisition frequency, and their origin (host or foreign DNA). I will use mutagenesis to evaluate the importance of conserved Csn2 residues for spacer acquisition. Lastly, I will determine if Csn2 impacts the selection of acquired spacers by interacting with specific protospacer sequences. The proposed research will uncover essential elements of SpyCas9 functioning by providing a greater understanding of the stringency of PAM recognition and by determining Csn2’s role during spacer acquisition in vivo. These findings have the potential to further fuel the CRISPR revolution in biomedicine, from the tracking of cell “memories” in pathogens and cancer cells, to the improvement of phage therapies, to the development of exquisitely precise gene editing systems. In doing, my findings will also ultimately transform patients’ lives.

项目摘要 CRISPR-Cas（规则间隔短回文重复序列和CRISPR相关基因） 作为原核生物的适应性免疫系统，通过提供对细菌病毒的保护来发挥作用 （二）。CRISPR基因座由散布有噬菌体DNA的短片段（间隔区）的重复序列组成。 CRISPR相关（cas）基因编码的蛋白质调节1）在细胞周期中获得新的间隔区， 噬菌体感染（“免疫”）和2）使用间隔区转录物来鉴定和降解互补的 外来DNA序列（原型间隔区）以防止感染（“免疫”）。我所提出的研究旨在更好地 了解酿脓链球菌II-A型CRISPR-Cas的调节组分的功能 系统（SpyCas 9）在基因编辑应用中几乎无处不在。 第一个目标涉及噬菌体DNA中发现的原型间隔区邻近基序（PAM）。经典SpyCas 9 PAM序列是“NGG”，但也可以看到非规范（非NGG）PAM序列。我收集了非NGG 从间隔区获取数据库中获得PAM，并鉴定出有效介导免疫的那些PAM。所有此类PAM 序列是“NAGG”因此，我假设NAGG PAM可以与Cas9相互作用，以介导显著的细胞凋亡。 免疫力为了验证这一点，我将确定每个核苷酸对免疫的重要性，以及免疫 数据库中未发现NAGG的潜力。我还将通过直接竞争比较AGG和NAGG PAM 在噬菌体靶向期间和通过所有可能的NAGG和AGG PAM的文库。最后，我将从生物化学的角度 在体外用DNA结合和切割测定表征NAGG PAM的作用。 第二个目标集中在神秘的SpyCas 9蛋白Csn 2上。其他SpyCas 9 Cas蛋白都很好- Csn 2已经被表征，但已知Csn 2仅对于体内间隔区获取是必需的。我调查了Csn 2对 关于在间隔区整合到靶细胞中之前从切割的原型间隔区体内获得间隔区的子步骤， CRISPR阵列。在这些阶段缺乏Csn 2会导致获得减少100倍。所以我 假设Csn 2通过在切割前与切割的原型间隔区相互作用来促进选择间隔区， 整合到CRISPR阵列中。我将测试WT和Δ csn 2菌株的间隔区采集，评估新的 长度、方向、获取频率和它们的来源（宿主或外源DNA）的间隔区。我会用 诱变以评估保守的Csn 2残基对于间隔区获取的重要性。最后，我将 确定Csn 2是否通过与特定的原型间隔区序列相互作用影响获得的间隔区的选择。 拟议的研究将通过提供更大的 了解PAM识别的严格性，并通过确定Csn 2在间隔区获取过程中的作用， vivo.这些发现有可能进一步推动生物医学中的CRISPR革命，从跟踪 病原体和癌细胞中的细胞“记忆”，噬菌体疗法的改进， 极其精确的基因编辑系统在这样做的过程中，我的发现也将最终改变患者的生活。