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

项目摘要 CRISPR-CAS(规则间隔短回文重复序列和CRISPR相关基因) 在原核生物中起适应性免疫系统的作用，提供对细菌病毒的保护 (噬菌体)。CRISPR基因座由穿插有噬菌体DNA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)新间隔区的获得 噬菌体感染(“免疫”)和2)使用间隔区转录本来识别和降解互补 防止感染的外来DNA序列(原核糖体)(“免疫”)。我提出的研究旨在更好地 了解化脓性链球菌II-A型CRISPR-CAS调节成分的功能 在基因编辑应用程序中几乎无处不在的SpyCas9系统。 第一个目的是关于在噬菌体DNA中发现的Protspacer相邻基序(PAM)。经典的SpyCas9 PAM序列是“NGG”，但也可以看到非规范的(非NGG)PAM序列。我收集了非NGG PAM从间隔区获取数据库，并确定了那些有效地介导免疫。所有这类PAM 序列为“NAGG”。因此，我假设NAGG PAM可以与Cas9相互作用，以调节显著的 豁免权。为了测试这一点，我将确定每个核苷酸对免疫的重要性，以及免疫 在数据库中看不到的NAGG的潜力。我还将通过直接竞争来比较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，我会在生物化学上 用DNA结合试验和切割试验鉴定NAGG PAM的体外作用。 第二个目标集中在神秘的SpyCas9蛋白Csn2上。其他SpyCas9 Cas蛋白都很好- Csn2具有特征性，但已知只有在体内获得间隔区是必不可少的。我调查了中国第二台的S撞击事件 在体内从裂解的蛋白间隔子获得间隔子的子步骤 CRISPR数组。在这些阶段中没有Csn2会导致获得量减少100倍。因此，我 假设Csn2通过与裂解的蛋白间隔子相互作用来促进选择间隔子 集成到CRISPR阵列中。我将测试从WT和Δcsn2毒株获得的间隔区，评估新的 长度、方向、获取频率及其来源(宿主或外源DNA)的间隔区。我会用 突变以评估保守的Csn2残基对于间隔区获取的重要性。最后，我会 确定Csn2是否通过与特定的Protspacer序列相互作用而影响获得的间隔子的选择。 拟议的研究将揭示SpyCas9功能的基本要素，通过提供更大的 理解PAM识别的严密性和确定Csn2在间隔区获得过程中的S作用 活着。这些发现有可能进一步推动CRISPR在生物医学领域的革命，从对 病原体和癌细胞中的细胞记忆，对噬菌体疗法的改进，对发展 精密的基因编辑系统。在这样做的过程中，我的发现也将最终改变患者的生活。

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.