Engineered Cas9 Nucleases with Single-Genomic-Site Precision for CYBB Correction

 描述（由申请人提供） II型CRISPR/Cas9系统正在彻底改变生物医学科学。这些可编程核酸酶促进在基因组内的特定位置处产生双链断裂，这通过用外源提供的供体DNA进行同源修复来促进靶向基因破坏或基因编辑。虽然现有的Cas9系统是强大的，但它们的混杂性对其在基因治疗应用中的实施构成了障碍，其中必须最小化或理想地消除对所治疗的基因组的不期望的附带损害。因此，进一步开发用于选择性识别和切割所需靶序列（并且仅该序列）的这种核酸酶平台是有必要的。 为了实现人类基因组内单位点核酸酶精确度的最终目标，我们提出开发Cas9和可编程DNA结合结构域（pDBD）之间的嵌合融合。我们已经建立并验证了一个工作原型，该原型比标准Cas9系统具有更高的精度，更大的活性和更广泛的序列靶向范围。在该提案中，我们概述了使用附加的pDBD来提高三种代表性的、经验证的Cas9直系同源物的精确度的实验：S.化脓链球菌和S. aureus Cas9（SpCas 9和SaCas 9;代表性的II-A型）和N.脑膜炎病毒Cas9（NmCas 9;代表性II-C型）。这些系统将应用于慢性肉芽肿病（CGD）的治疗基因校正。在目标1中，我们将优化我们建立的SpCas 9-pDBD融合体的特征，以创建嵌合系统，该嵌合系统需要额外的靶切割许可阶段，并通过药物依赖性二聚化系统并入外源调节。在目标2中，我们将把Cas9-pDBD融合体的优势扩展到更紧凑的NmCas 9和SaCas 9中，并确定II-A型和II-C型Cas9-pDBD融合体之间基本设计原理的相似性和差异。在目标3中，我们将应用我们的Cas9-pDBD系统来精确和有效地校正造血干细胞中与CGD相关的X连锁缺陷，以建立针对这种毁灭性疾病的基于基因校正的自体干细胞疗法。 最终，拟议的研究有望产生基因组编辑酶，这些酶表现出在临床基因治疗和干细胞工程中安全有效应用所需的特异性，我们将通过创建基于细胞的CGD基因治疗来证明这一点。

 DESCRIPTION (provided by applicant) Type II CRISPR/Cas9 systems are revolutionizing biomedical science. These programmable nucleases facilitate the creation of a double strand break at a specific location within a genome, which promotes targeted gene disruption or gene editing through homologous repair with an exogenously supplied donor DNA. While existing Cas9 systems are powerful, their promiscuity presents a barrier to their implementation in gene therapy applications, where undesired collateral damage to the treated genome must be minimized or, ideally, eliminated. Consequently, further development of this nuclease platform for the selective recognition and cleavage of a desired target sequence (and only that sequence) is warranted. To achieve the ultimate goal of single-site nuclease precision within the human genome, we propose to develop a chimeric fusion between Cas9 and a programmable DNA-binding domain (pDBD). We have established and validated a working prototype that has improved precision, greater activity, and a broader sequence targeting range than the standard Cas9 system. In this proposal, we outline experiments to use appended pDBDs to improve precision of three representative, validated Cas9 orthologs: S. pyogenes and S. aureus Cas9 (SpCas9 & SaCas9; representative Type II-A) and N. meningitidis Cas9 (NmCas9; representative Type II-C). These systems will be applied to the therapeutic gene correction of chronic granulomatous disease (CGD). In Aim 1, we will optimize the characteristics of our established SpCas9-pDBD fusions to create a chimeric system that requires an additional stage of licensing for target cleavage and incorporates exogenous regulation through a drug-dependent dimerization system. In Aim 2, we will extend the advantages of Cas9-pDBD fusions into the more compact NmCas9 and SaCas9, and identify the similarities and differences in essential design principles between Type II-A and Type II-C Cas9-pDBD fusions. In Aim 3, we will apply our Cas9-pDBD system to the precise and efficient correction in hematopoietic stem cells of X-linked defects that are associated with CGD to establish a gene correction-based autologous stem cell therapy for this devastating disease. Ultimately, the proposed research promises to yield genome-editing enzymes that exhibit the specificity required for safe, effective application in clinical gene therapy and stem cell engineering, which we will demonstrate by creating a cell-based gene therapy for CGD.