FFRDC MOONSHOT PORTFOLIO TASK ORDER - CRISPR gRNA Reagents for NGCMs

FFRDC MOONSHOT 组合任务订单 - 用于 NGCM 的 CRISPR gRNA 试剂

• 批准号: 10716701
• 负责人: CHRISTINE SIEMON
• 金额: $ 41.33万
• 依托单位: LEIDOS BIOMEDICAL RESEARCH, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-31 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10716701
• 关键词: Address; Affect; Amino Acids; BCAR1 gene; Bacteria; Base Pairing; Binding; Biological; Cancer Center; Cancer Etiology; Cancer Model; Cancer cell line; Caspase; Cell Differentiation process; Cell Line; Cell Proliferation; Cells; Chromosome Structures; Clinical; Coculture Techniques; Code; Communities; Complex; Culture Media; DNA; DNA Binding; DNA Sequence; DNA-Binding Proteins; Data; Deoxyribonucleases; Disease; Enzymes; Epidermal Growth Factor; Epigenetic Process; Epithelial Cells; Ethnic group; Extracellular Matrix; Fibroblasts; Formulation; Genes; Genetic; Genetic Transcription; Genome; Genomics; Goals; Guide RNA; Human; Human Genome; In Vitro; Institution; Intellectual Property; International; Laboratories; Light; Location; Malignant Childhood Neoplasm; Malignant Neoplasms; Mediating; Mesenchyme; Methodology; Methods; Modeling; Modification; Molecular; Mus; Mutate; Mutation; Nucleosomes; Office of Cancer Genomics; Open Reading Frames; Organ; Organoids; Pathology; Patients; Phenotype; Population; Proliferating; Promoter Regions; Proteins; Protocols documentation; RNA; RNA Binding; Reagent; Request for Proposals; Research; Research Personnel; Rho-associated kinase; Site; Small Intestines; Specificity; Streptococcus pyogenes; Structure; System; Technology; Tissues; Transcription Coactivator; Transcription Initiation Site; Transcriptional Activation; Treatment outcome; Tumor Subtype; Vascular blood supply; Virus Diseases; analytical method; cancer genome; cancer genomics; cell type; epigenome; epithelial stem cell; established cell line; flexibility; genome editing; in vivo; insertion/deletion mutation; keratinocyte; kinase inhibitor; mammalian genome; monolayer; nerve supply; new therapeutic target; next generation; novel; nuclease; ovarian neoplasm; promoter; rare cancer; response; stem cells; stem-like cell; three dimensional structure; transcriptome; treatment response; tumor; whole genome

Next Generation Cancer Models (NGCMs) Most 2-dimentional cancer cell lines currently used to study the etiology of cancer, e.g. mechanism, treatment response, etc. inadequately model its genetic, epigenetic, and phenotypic complexity. Many lack molecular characterization of the original tumor’s genome, epigenome and transcriptome (and the case-matched normal DNA) as well as the clinical presentation, treatment, and outcome data. In addition, cell lines from certain subtypes (defined by pathology or molecular features) or populations, as well as rare and pediatric cancers, are underrepresented or not available. Reasonably robust protocols to establish the NGCMs from a number of tumor subtypes have been developed over the past several years. Organoid, conditionally reprogrammed cells (CRC), modified versions of those methods, and unique media formulations can be used to establish models, which appear to address current gaps in cancer models. Some protocols even allow co-culture of tumor and stroma tissues. Organoid culture methods were first created using stem cells from the mouse small intestine. The models usually consist of two or more cell types and develop structures that resemble in vivo organs, but lack innervation, blood supply, and mesenchyme. These three-dimensional structures are grown from epithelial stem cells and are amenable to expansion in culture under appropriate conditions. Cultures are grown in exogenous extracellular matrix using media that contain components that drive cell proliferation and differentiation, such as Noggin, R-spondin, Wnt, and epidermal growth factor. The method has been adapted to propagate other cell and tumor subtypes for many months which appear to be representative of the original tumors. Research is ongoing to determine if the NGCMs are immortal, genomically stable etc. CRC methodology was pioneered using human keratinocytes and subsequently expanded to other epithelial cell types. Propagation of primary or tumor human cells requires Rho kinase inhibitor and irradiated mouse fibroblast feeder cells. The CRCs proliferate indefinitely as stem-like cells but appear to maintain the ability to differentiate. Although they are typically cultured as monolayers, CRCs can also develop into organ-like three-dimensional structures when grown in a supporting matrix. New media formulations have also been very successful in establishing models from tumor types historically difficult to propagate in culture. As opposed to standard culture media that contain on average 40-50 ingredients, these new media have over 80 components. For example, using complex medium to establish cell lines from a diverse array of ovarian tumors, results in cultures that retain most of the genomic and molecular features of the tissue from which they originated. The NCI Office of Cancer Genomics (OCG), Center for Cancer Genomics, together with international institutions, established a consortium, the Human Cancer Models Initiative (HCMI) whole goal is to make available to the scientific community large numbers of the in vitro NGCMs from many tumor subtypes and patient ethnic groups that are not encumbered with excessive intellectual property (IP) constraints. The conditions used for propagation are freely available. Genomic Pertubagens Genetic methods are successful in exploring the mechanisms of disease and identifying novel therapeutic targets. The clustered regularly interspersed short palindromic repeats (CRISPRs) gene editing, a bacterial defense system against virus infection, is the basis of the newest mammalian genome editing technology. It consists of a DNA binding protein, usually a nuclease, and RNA(s), termed guide RNA (gRNA), that allows the DNA cutting to occur at the desired locus, though not necessarily in an exact location. One version of the method allows researchers to permanently modify genes in living cells. Since the discovery of this biological modification mechanism, research identified this defense system many in bacterial species and the nuclease protein can be quite distinct. In addition, a number of laboratories created “dead” nucleases which bind the DNA, but do not cut it and thereby can be used for transcriptional activation and inhibition without changing the genome. Each bacterium which uses the “CRISPR” system has its own nuclease and other requirements for function. The most commonly used enzyme for mammalian gene editing is Streptococcus pyogenes’ Cas9 (SpCas9) whose open reading frame (ORF) is 1368 amino acids. When the Cas9 complex cuts DNA, it cuts both strands at the same place, leaving ‘blunt ends’ that often undergo changes/mutations as they are rejoined. Cpf1/Cas12a cuts DNA differently from Cas9 and is about 300 amino acids smaller. The Cpf1 system provides flexibility in choosing target sites. Similarly to Cas9, the Cpf1 complex must first attach to the protospacer adjacent motif (PAM, 2-6 base pair DNA sequence) and targets must be chosen that are adjacent to naturally occurring PAM sequences. The Cpf1 complex recognizes very different PAM sequences from Cas9. The Cpf1 complex cuts in the DNA helix strands are offset, leaving short overhangs at the ends. This is expected to help with precise insertion, allowing researchers to integrate a piece of DNA more efficiently and accurately. Cpf1 cuts far away from the recognition site, meaning that even if the targeted gene becomes mutated at the cut site, it can likely still be re-cut, allowing multiple opportunities for correct editing to occur. The gRNAs are very important for the specificity of the Cas binding. They are made up of 2 components, the sequence to which they will bind within the genome and protospacer adjacent motif (PAM). These PAMs are caspase dependent and affect specificity. A nuclease can be modified to reduce the PAM specificity/requirement. In addition, the nuclease editing of a gene provides more flexibility for gRNA binding. In summary, the NGCMs be utilized in research that will shed light on cancer mechanisms, response to novel treatments and even serve as patient avatars. Therefore, the experimental protocols to manipulate the NGCMs, the technologies and analytical methods need to be optimized for these applications. The goal of this RFP is to generate validated human editing reagents for all transcribed genes, i.e. “whole genome”, through a single construct of nuclease and gRNA(s).

下一代癌症模型 (NGCM) 目前大多数二维癌细胞系用于研究癌症的病因学，例如癌细胞。机制、治疗反应等不足以对其遗传、表观遗传和表型复杂性进行建模。许多缺乏原始肿瘤基因组、表观基因组和转录组（以及病例匹配的正常 DNA）的分子特征以及临床表现、治疗和结果数据。此外，来自某些亚型（由病理学或分子特征定义）或群体的细胞系，以及罕见和儿童癌症的细胞系代表性不足或不可用。 在过去的几年里，已经开发出相当稳健的方案来从多种肿瘤亚型中建立 NGCM。类器官、条件重编程细胞（CRC）、这些方法的修改版本以及独特的培养基配方可用于建立模型，这似乎解决了癌症模型中当前的空白。一些方案甚至允许肿瘤和基质组织的共培养。 类器官培养方法首先是使用小鼠小肠干细胞创建的。这些模型通常由两种或多种细胞类型组成，并形成类似于体内器官的结构，但缺乏神经支配、血液供应和间质。这些三维结构由上皮干细胞生长而成，并且适合在适当条件下在培养物中扩增。使用含有驱动细胞增殖和分化的成分（例如 Noggin、R-spondin、Wnt 和表皮生长因子）的培养基在外源细胞外基质中培养培养物。该方法已适应于繁殖其他细胞和肿瘤亚型数月，这些细胞和肿瘤亚型似乎代表了原始肿瘤。正在进行研究以确定 NGCM 是否永生、基因组稳定等。 CRC 方法首先使用人类角质形成细胞，随后扩展到其他上皮细胞类型。原代细胞或肿瘤人类细胞的增殖需要 Rho 激酶抑制剂和经过辐射的小鼠成纤维细胞饲养细胞。 CRC 作为干细胞样细胞无限增殖，但似乎保持分化能力。尽管CRC通常以单层培养，但当在支持基质中生长时，CRC也可以发育成器官样的三维结构。 新的培养基配方在建立历来难以在培养物中繁殖的肿瘤类型模型方面也非常成功。与平均含有 40-50 种成分的标准培养基不同，这些新培养基含有 80 多种成分。例如，使用复合培养基从多种卵巢肿瘤中建立细胞系，产生的培养物保留了其起源组织的大部分基因组和分子特征。 NCI 癌症基因组学办公室 (OCG)、癌症基因组学中心与国际机构共同建立了一个联盟，即人类癌症模型计划 (HCMI)，其总体目标是向科学界提供大量来自许多肿瘤亚型和患者种族的体外 NGCM，且不受过多的知识产权 (IP) 限制。用于传播的条件是免费提供的。 基因组影响因素 遗传学方法成功地探索了疾病的机制并确定了新的治疗靶点。成簇规律散布的短回文重复序列（CRISPR）基因编辑是一种针对病毒感染的细菌防御系统，是最新的哺乳动物基因组编辑技术的基础。它由 DNA 结合蛋白（通常是核酸酶）和 RNA（称为向导 RNA (gRNA)）组成，允许 DNA 切割发生在所需的位点，但不一定在精确的位置。该方法的一种版本允许研究人员永久修改活细胞中的基因。自从发现这种生物修饰机制以来，研究发现这种防御系统在许多细菌物种中与核酸酶蛋白可以相当不同。此外，许多实验室创造了“死”核酸酶，它们结合 DNA，但不切割 DNA，因此可用于转录激活和抑制而不改变基因组。 每种使用“CRISPR”系统的细菌都有自己的核酸酶和其他功能要求。最常用的哺乳动物基因编辑酶是化脓性链球菌的 Cas9 (SpCas9)，其开放阅读框 (ORF) 为 1368 个氨基酸。当 Cas9 复合体切割 DNA 时，它会在同一位置切割两条链，留下“钝端”，这些“钝端”在重新连接时经常会发生变化/突变。 Cpf1/Cas12a 切割 DNA 的方式与 Cas9 不同，大约小 300 个氨基酸。 Cpf1 系统提供了选择目标位点的灵活性。与 Cas9 类似，Cpf1 复合体必须首先连接到原型间隔子相邻基序（PAM，2-6 个碱基对 DNA 序列），并且必须选择与天然存在的 PAM 序列相邻的靶标。 Cpf1 复合体识别与 Cas9 非常不同的 PAM 序列。 DNA 螺旋链中的 Cpf1 复合体切口被抵消，在末端留下短突出端。这有望有助于精确插入，使研究人员能够更有效、更准确地整合 DNA 片段。 Cpf1 的切割距离识别位点很远，这意味着即使目标基因在切割位点发生突变，它仍然可能被重新切割，从而提供多次正确编辑的机会。 gRNA 对于 Cas 结合的特异性非常重要。它们由 2 个组件组成，即它们在基因组内结合的序列和原型间隔子相邻基序 (PAM)。这些 PAM 依赖于半胱天冬酶并影响特异性。可以修改核酸酶以降低 PAM 特异性/要求。此外，基因的核酸酶编辑为 gRNA 结合提供了更大的灵活性。 总之，NGCM 可用于研究，以阐明癌症机制、对新疗法的反应，甚至充当患者的化身。因此，操作 NGCM 的实验方案、技术和分析方法需要针对这些应用进行优化。该 RFP 的目标是通过核酸酶和 gRNA 的单一构建体为所有转录基因（即“全基因组”）生成经过验证的人类编辑试剂。