Design and Development of Ligand-Responsive CRISPR-Cas Enzymes

配体响应性 CRISPR-Cas 酶的设计和开发

• 批准号: 10388925
• 负责人: Brady Fletcher Cress
• 金额: $ 0.25万
• 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/10388925
• 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.

项目总结 用于基因组和转录组操作的CRISPR-Cas酶的发现和再利用 深刻地影响了实验设计和研究的速度、广度和深度 医学。对不同类型CRISPR-CAS酶的研究继续发现新的化学物质 使这些蛋白质非常适合于新型研究和研究的机制和生物学作用 治疗应用。例如，最近有几个Cas9同源基因被证明可以结合和切割 一种RNA引导的、非依赖于蛋白质的邻接基序(PAM)的方式，使蛋白质能够在体内被抑制 通过靶向的mRNA结合表达，甚至抑制RNA噬菌体感染。几个机箱12a 最近发现，同源基因一旦被激活，就会作为非特异性单链DNA(SsDNA)核酸酶 RNA引导的靶DNA结合，这一特性可用于干扰人类致病病毒的生命周期 单链DNA细小病毒或微生物单链DNA噬菌体。因此，这些CRISPR-CAS酶继续有希望 令人兴奋的、创新的RNA和单链DNA靶向应用，超越了其现有的有效实施 作为基因组编辑。然而，在将这些酶用作医疗工具之前，谨慎的做法是设计和测试 它们的核酸修饰和结合活性可以被快速激活或 停用以防止不需要的编辑。拟议研究的目标是发展变构 受调控的CRISPR-CAS酶能够精确地时空控制下一代基因组和 转录组修饰。在第一个目标中，变构敏感部位将在一个 一组医学上有用的Cas12a和RNA靶向Cas9同源基因，产生了一组新的CRISPR-CA 由局部给药4-羟基三苯氧胺(4-HT)或雷帕霉素控制的酶。在第二个 目的，我们将扩大能够控制CRISPR的配体和代谢产物的化学多样性。 移植天然传感器蛋白配体结合调节结构域的CAS酶活性 进入Cas9和Cas12a同源基因的变构敏感部位。第三个目标是变构CRISPR-Cas分子 将部署记录器，以量化遗传上的异种细胞群体的代谢失调 编码可通过深度测序检索的单细胞代谢特征。这些目标的成功将设定 CRISPR-Cas酶自动感知和响应动态图谱的发展阶段 定义的化学线索组合，便于安全地部署用于治疗的智能核酸编辑器 应用程序以及使用传统报告器或通过 基因组编码记录。加州大学伯克利分校提供协作性、合议性、跨学科和科学的 严格的环境，有利于高效的博士后科学和专业培训； 其成熟的、世界级的调查人员拥有技术专长，以补充 杜德纳实验室，并确保拟议的研究金培训计划取得成功。