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 DNA修复酶 摘要 目前用于人类基因组编辑的工具包可以通过探索大幅扩展 尚未鉴定的新CRISPR-Cas蛋白，并通过开发 酶工具，以实现DNA断裂的受控修复。我们的目标是推动科学和 发现和利用新的CRISPR-CA及其相关基因的基因组编辑技术 通过生物信息学和生物化学相结合的方式建立系统。 首先，我们将扩展CRISPR-CasX和-Casy序列的多样性，并分析相关的 参与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座位的组织和功能成分，并进行 对重组系统进行试验，以建立行动机制并使 在哺乳动物细胞中进行测试。第二，我们将确定CRISPR-CasZ蛋白的机制， 一个新发现的RNA引导酶超家族。这些蛋白质非常小， 多样性，并具有目前未定义的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和 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。将根据蛋白质识别新的基因变种 家族关系、功能预测和基因组邻里关系。我们将建立检测方法 测定包括DNA和RNA结合、解离和切割在内的分子活性。 总而言之，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， 有助于基因组监测和操作的酶和转录本 微生物世界。这将带来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.