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

 描述(由申请人提供) 第二类CRISPR/CAS9系统正在给生物医学科学带来革命性的变化。这些可编程的核酸酶有助于在基因组的特定位置产生双链断裂，通过与外源提供的供体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系统功能强大，但它们的混杂对其在基因治疗应用中的实施构成了障碍，在基因治疗应用中，必须最大限度地减少或理想情况下消除对已处理基因组的不必要的附带损害。因此，有必要进一步开发该核酸酶平台，以选择性识别和切割所需的靶序列(并且只有该序列)。为了在人类基因组中实现单位点核酸酶的精确度的最终目标，我们建议在Cas9和可编程DNA结合结构域(PDBD)之间建立嵌合融合。我们已经建立并验证了一个工作原型，它比标准的Cas9系统具有更高的精度、更大的活性和更宽的序列靶向范围。在这份提案中，我们概述了使用附加的pDBDS来提高三个具有代表性的、经过验证的Cas9同源基因的精确度的实验：化脓性链球菌和金黄色葡萄球菌Cas9(SpCas9和SaCas9；代表性的类型II-A)和脑膜炎奈瑟氏菌Cas9(NmCas9；代表性的类型II-C)。这些系统将应用于慢性肉芽肿性疾病(CGD)的治疗性基因校正。在目标1中，我们将优化我们已经建立的SpCas9-pDBD融合的特征，以创建一个嵌合系统，该系统需要额外的靶标切割许可阶段，并通过药物依赖的二聚系统纳入外源调节。在目标2中，我们将把Cas9-pDBD融合的优势扩展到更紧凑的NmCas9和SaCas9，并确定II-A型和II-C型Cas9-pDBD融合在基本设计原则上的异同。在目标3中，我们将应用我们的Cas9-pDBD系统精确而有效地纠正与CGD相关的X连锁缺陷的造血干细胞，以建立一种基于基因纠正的自体干细胞疗法来治疗这种毁灭性的疾病。最终，这项拟议的研究有望生产出显示出安全、有效应用于临床基因治疗和干细胞工程所需的特异性的基因组编辑酶，我们将通过创建一种基于细胞的CGD基因疗法来展示这一点。

 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.