Design and Development of Ligand-ResponsiveCRISPR-Cas Enzymes

配体响应性CRISPR-Cas酶的设计和开发

• 批准号: 10023929
• 负责人: Brady Fletcher Cress
• 金额: $ 7.05万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10023929
• 关键词: 4-Hydroxy-Tamoxifen; Allosteric Regulation; Analytical Chemistry; Attenuated; BCAR1 gene; Bacteriophages; Bar Codes; Basic Science; Binding; Biological; Biological Process; Cells; Chemicals; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Cues; Cyclic GMP; DNA; DNA sequencing; Deoxyribonucleases; Detection; Development; Diagnostic; Disease; Dose; Engineering; Ensure; Environment; Enzymes; Event; Experimental Designs; Feedback; Fellowship; Gases; Gene Expression; Generations; Genetic Transcription; Genome; Genomic DNA; Genomics; Guide RNA; Harvest; Human; Image; In Situ; Infection; Investigation; Libraries; Life Cycle Stages; Ligand Binding; Ligand Binding Domain; Ligands; Light; Location; Longitudinal Studies; Maps; Measurement; Medical; Messenger RNA; Metabolic; Modification; Molecular; Nature; Nucleic Acid Binding; Nucleic Acids; Orthologous Gene; Output; Parvovirus; Pathogenesis; Pathogenicity; Peptides; Physiological; Population; Process; Production; Property; Proteins; RNA; RNA Phages; Recording of previous events; Regulation; Reporter; Reporting; Repression; Research; Research Personnel; Resolution; Role; Science; Signal Transduction; Single-Stranded DNA; Sirolimus; Site; Soluble Guanylate Cyclase; Source; Specificity; Structure; Technical Expertise; Technology; Testing; Therapeutic; Training; Transcription Repressor; Transplantation; Validation; Variant; Work; analog; base; cell type; deep sequencing; design; empowered; enzyme activity; epigenome editing; genome editing; improved; in vivo; innovation; knock-down; member; metabolic abnormality assessment; metabolic profile; microbial; next generation; novel; nuclease; prevent; programs; promoter; protein expression; response; screening; sensor; single cell sequencing; small hairpin RNA; small molecule; spatiotemporal; success; synthetic biology; temporal measurement; therapeutic genome editing; tool; transcriptome

PROJECT SUMMARY The discovery and repurposing of CRISPR-Cas enzymes for genome and transcriptome manipulation has profoundly impacted the pace, breadth, and depth of experimental design and investigation in the biological and medical sciences. The search for distinct types of CRISPR-Cas enzymes continues to uncover novel chemical mechanisms and biological roles that make these proteins well suited for new types of investigative and therapeutic applications. For example, several Cas9 orthologs were recently shown to bind and cleave RNA in an RNA-guided, protospacer adjacent motif (PAM)-independent manner, enabling in vivo repression of protein expression through targeted mRNA binding and even inhibiting RNA bacteriophage infection. Several Cas12a orthologs were recently found to act as non-specific single-stranded DNA (ssDNA) nucleases once activated by RNA-guided binding of target DNA, a property that could be used to interfere with life cycles of human pathogenic ssDNA parvoviruses or microbial ssDNA bacteriophage. Thus, these CRISPR-Cas enzymes continue to promise exciting, innovative RNA- and ssDNA-targeting applications beyond their established impactful implementation as genome editors. Before deploying these enzymes as medical tools, however, it is prudent to design and test mechanisms through which their nucleic acid-modifying and -binding activities can be rapidly activated or inactivated to prevent undesired editing. The objective of the proposed research is to develop allosterically regulated CRISPR-Cas enzymes to enable precise spatiotemporal control over next-generation genome and transcriptome modification. In the first aim, allosterically sensitive sites will be systematically mapped within a set of medically useful Cas12a and RNA-targeting Cas9 orthologs, yielding a panel of new CRISPR-Cas enzymes that are controlled by local administration of 4-hydroxytamoxifen (4-HT) or rapamycin. In the second aim, we will expand the chemical diversity of ligands and metabolites capable of exerting control over CRISPR- Cas enzyme activity by transplanting ligand-binding regulatory domains harvested from natural sensor proteins into allosterically sensitive sites in Cas9 and Cas12a orthologs. In the third aim, allosteric CRISPR-Cas molecular recorders will be deployed to quantify metabolic dysregulation across a heterogenous cell population, genetically encoding single cell metabolic profiles that are retrievable by deep sequencing. Success of these aims will set the stage for development of CRISPR-Cas enzymes that automatically sense and respond to dynamic profiles of defined chemical cue combinations, facilitating safe deployment of smart nucleic acid editors for therapeutic applications and for longitudinal reporting of intracellular ligand states using traditional reporters or through genome-encoded recording. UC Berkeley offers a collaborative, collegial, interdisciplinary, and scientifically rigorous environment that is conducive to highly effective postdoctoral scientific and professional training, and its established, world-class investigators possess the technical expertise to complement that provided in the Doudna lab and to ensure the success of the proposed fellowship training plan.

项目摘要 用于基因组和转录组操纵的CRISPR-Cas酶的发现和再利用已经成为一种新的技术。 深刻地影响了生物学实验设计和研究的速度、广度和深度， 医学科学对不同类型的CRISPR-Cas酶的研究继续揭示新的化学物质 机制和生物学作用，使这些蛋白质非常适合于新类型的调查， 治疗应用。例如，最近显示几种Cas9直向同源物结合并切割RNA， 一种RNA引导的、不依赖于前间区序列邻近基序（PAM）的方式，能够在体内抑制蛋白质 通过靶向mRNA结合甚至抑制RNA噬菌体感染来表达。几个Cas 12 a 最近发现直系同源物一旦被激活就充当非特异性单链DNA（ssDNA）核酸酶。 RNA引导的靶DNA结合，这是一种可用于干扰人类病原体生命周期的特性 ssDNA细小病毒或微生物ssDNA噬菌体。因此，这些CRISPR-Cas酶继续承诺 令人兴奋的创新RNA和ssDNA靶向应用，超越其既定的影响力实施 作为基因组编辑者。然而，在将这些酶用作医疗工具之前， 通过其核酸修饰和结合活性可以被快速激活的机制，或 禁用以防止不希望的编辑。拟议研究的目的是发展变构 调控CRISPR-Cas酶，以实现对下一代基因组的精确时空控制， 转录组修饰在第一个目标中，变构敏感位点将系统地定位在 一组医学上有用的Cas 12 a和RNA靶向Cas9直系同源物，产生一组新的CRISPR-Cas 通过局部施用4-羟基他莫昔芬（4-HT）或雷帕霉素控制的酶。在第二 我们将扩大能够控制CRISPR的配体和代谢物的化学多样性。 通过移植从天然传感器蛋白收获的配体结合调节结构域的Cas酶活性 插入Cas9和Cas 12 a直系同源物中的变构敏感位点。在第三个目标中，变构CRISPR-Cas分子 记录器将被部署来量化跨异质细胞群的代谢失调，遗传学上， 编码可通过深度测序检索的单细胞代谢谱。这些目标的实现将为 CRISPR-Cas酶的开发阶段，其自动感测和响应动态概况 定义的化学提示组合，促进智能核酸编辑器的安全部署，用于治疗 应用和用于使用传统报告物或通过 基因组编码记录加州大学伯克利分校提供了一个协作，合议，跨学科，科学 严格的环境，有利于高效的博士后科学和专业培训， 其既定的，世界一流的调查人员拥有的技术专长，以补充提供的， 杜德纳实验室，并确保拟议的奖学金培训计划的成功。