Expanding CRISPR-Cas editing technology through exploration of novel Cas proteins and DNA repair systems

通过探索新型 Cas 蛋白和 DNA 修复系统扩​​展 CRISPR-Cas 编辑技术

• 批准号: 10459340
• 负责人: Jillian Banfield
• 金额: $ 39.25万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-24 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10459340
• 关键词: BCAR1 gene; Base Sequence; Biochemical; Biochemistry; Bioinformatics; Biological Assay; Catalytic Domain; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Binding; DNA Repair; DNA Repair Enzymes; Data Set; Development; Enzymes; Genes; Genetic; Genome; Genomics; Guide RNA; Human Genome; Investigation; Mammalian Cell; Metagenomics; Microbe; Molecular; Neighborhoods; Open Reading Frames; Pathway interactions; Polymerase; Productivity; Property; Protein Family; Proteins; RNA; RNA Binding; Radiation; Research Personnel; Science; Small RNA; System; Technology; Testing; Transcript; Trees; Variant; Work; adaptive immunity; base; cofactor; experimental study; fundamental research; genetic variant; genome editing; genomic locus; genomic tools; helicase; insight; metagenomic sequencing; microbial; molecular sequence database; novel; nuclease; programs; recombinase; reconstitution; success; tool; transcriptomics

Expanding genome editing tools through exploration of new CRISPR-Cas proteins and DNA repair enzymes ABSTRACT The current toolkit for human genome editing could be dramatically expanded through exploration of new CRISPR-Cas proteins that have yet to be characterized, and through the development of enzymatic tools to enable controlled repair of DNA breaks. We aim to advance the science and technology of genome editing by discovering and harnessing new CRISPR-Cas and related systems through a combination of bioinformatics and biochemistry. First, we will expand the diversity of CRISPR-CasX and -CasY sequences and analyze associated proteins, cofactors and small RNAs that contribute to the function of CasX and CasY enzymes. We will define the organization and functional components of these genomic loci, and conduct experiments with reconstituted systems that will establish mechanisms of action and enable testing in mammalian cells. Second, we will determine the mechanism of CRISPR-CasZ proteins, a newly discovered superfamily of RNA-guided enzymes. These proteins are exceptionally small, diverse, and have currently undefined functionalities for manipulating genomic sequences. Third, we will use protein family analyses that leverage huge datasets from uncultivated microbes and transcriptomics to discover and evaluate new enzymes for DNA manipulation, including helicases, nucleases, polymerases and recombinases. New gene variants will be identified based on protein family affiliation, functional predictions and genomic neighborhoods. We will establish assays for determining molecular activities including DNA and RNA binding, unwinding and cleavage. Together, the results to be obtained from these aims will lead to new insights into the functions of known proteins within CRISPR-Cas systems, and will likely uncover new kinds of proteins, enzymes and transcripts that contribute to genome surveillance and manipulation across the microbial world. This will lead to a much larger toolbox for genome editing than is currently available, enabling researchers and clinicians to quickly optimize technologies according to their needs.

通过探索新的CRISPR-Cas蛋白质来扩展基因组编辑工具， DNA修复酶 摘要 通过探索，目前用于人类基因组编辑的工具包可以大幅扩展 新的CRISPR-Cas蛋白尚未被表征，通过开发 酶促工具，以实现DNA断裂的受控修复。我们的目标是推动科学和 通过发现和利用新的CRISPR-Cas和相关的基因组编辑技术 系统通过生物信息学和生物化学的结合。 首先，我们将扩大CRISPR-CasX和-CasY序列的多样性，并分析相关的基因。 蛋白质，辅因子和小RNA，有助于CasX和CasY酶的功能。 我们将定义这些基因组位点的组织和功能成分，并进行 用重组系统进行实验，建立作用机制， 在哺乳动物细胞中进行测试。其次，我们将确定CRISPR-CasZ蛋白的作用机制， 一个新发现的RNA引导酶超家族这些蛋白质非常小， 多种多样，并且目前还没有定义用于操纵基因组序列的功能。第三、 我们将使用蛋白质家族分析，利用来自未培养微生物的巨大数据集， 转录组学用于发现和评估用于DNA操作的新酶，包括解旋酶， 核酸酶、聚合酶和重组酶。新的基因变异将根据蛋白质 家庭关系、功能预测和基因组邻近区。我们将建立分析， 确定分子活性，包括DNA和RNA结合、解旋和切割。 总之，从这些目标中获得的结果将导致对 CRISPR-Cas系统中的已知蛋白质，并可能发现新的蛋白质种类， 酶和转录物，有助于基因组监视和操纵， 微生物世界这将导致一个比目前更大的基因组编辑工具箱 使研究人员和临床医生能够根据他们的需求快速优化技术。 需求

项目成果

RNA language models predict mutations that improve RNA function.

RNA 语言模型预测可改善 RNA 功能的突变。

• DOI: 10.1101/2024.04.05.588317
• 发表时间: 2024
• 期刊: bioRxiv : the preprint server for biology
• 作者: Shulgina,Yekaterina;Trinidad,MarenaI;Langeberg,ConnerJ;Nisonoff,Hunter;Chithrananda,Seyone;Skopintsev,Petr;Nissley,AmosJ;Patel,Jaymin;Boger,RonS;Shi,Honglue;Yoon,PeterH;Doherty,ErinE;Pande,Tara;Iyer,AdityaM;Doudna,Jenni
• 通讯作者: Doudna,Jenni

Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes.

• DOI: 10.1093/nar/gkad1053
• 发表时间: 2023-12-11
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Yoon PH;Skopintsev P;Shi H;Chen L;Adler BA;Al-Shimary M;Craig RJ;Loi KJ;DeTurk EC;Li Z;Amerasekera J;Trinidad M;Nisonoff H;Chen K;Lahiri A;Boger R;Jacobsen S;Banfield JF;Doudna JA
• 通讯作者: Doudna JA