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

通过探索新的 CRISPR-Cas 蛋白和扩展基因组编辑工具 DNA修复酶 摘要 当前的人类基因组编辑工具包可以通过探索得到极大的扩展 尚未表征的新 CRISPR-Cas 蛋白，并通过开发 能够控制 DNA 断裂修复的酶工具。我们的目标是推进科学和 通过发现和利用新的 CRISPR-Cas 及相关技术进行基因组编辑技术 生物信息学和生物化学相结合的系统。 首先，我们将扩展 CRISPR-CasX 和 -CasY 序列的多样性并分析相关的 有助于 CasX 和 CasY 酶功能的蛋白质、辅因子和小 RNA。 我们将定义这些基因组位点的组织和功能组件，并进行 重组系统的实验将建立行动机制并实现 在哺乳动物细胞中进行测试。其次，我们将确定 CRISPR-CasZ 蛋白的机制， 新发现的RNA引导酶超家族。这些蛋白质非常小， 多样化，并且目前具有未定义的操作基因组序列的功能。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。将根据蛋白质鉴定新的基因变异 家族归属、功能预测和基因组邻域。我们将建立检测 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带来对功能的新见解。 CRISPR-Cas系统中已知的蛋白质，并且可能会发现新的蛋白质种类， 有助于整个基因组监视和操作的酶和转录本 微生物世界。这将带来比目前更大的基因组编辑工具箱 可用，使研究人员和临床医生能够根据他们的情况快速优化技术 需要。

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.