Supplementary Information to publication "CASCADE-Cas3 Enables Highly Efficient Genome Engineering in <i>Streptomyces</i> Species"

Supplementary Information for manuscript "CASCADE-Cas3 Enables Highly Efficient Genome Engineering in <i>Streptomyces </i>Species". Includes additional data related to genome mining, bioinformatics, as well as plasmids and primers used in the study. Abstract: Type I CRISPR systems are widespread in bacteria and archaea. Compared to more widely applied type II systems, type I systems differ in the multi-effector CASCADE needed for crRNA processing and target recognition, as well as the processive nature of the hallmark nuclease Cas3. Given the widespread nature of type I systems, the processive nature of Cas3 and the recombinogenic overhangs created by Cas3, we hypothesized that Cas3 would be uniquely positioned to enable efficient genome engineering in streptomycetes. Here, we report a new type I based CRISPR genome engineering tool for streptomycetes. The plasmid system, called pCRISPR-Cas3, utilizes a compact type I-C CRISPR system and enables highly efficient genome engineering. pCRISPR-Cas3 outperforms pCRISPR-Cas9 and facilitates targeted and random sized deletions. Furthermore we demonstrate its ability to effectively perform substitutions of large genomic regions such as biosynthetic gene clusters. Without additional modifications, pCRISPR-Cas3 enabled genome engineering in several <i>Streptomyces </i>species at high efficiencies.论文《"CASCADE-Cas3可高效实现链霉菌属（Streptomyces）物种的基因组工程编辑"》补充材料。包含本研究涉及的基因组挖掘、生物信息学相关附加数据，以及研究中使用的质粒与引物。 **摘要**：I型成簇规律间隔短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR）系统广泛分布于细菌与古菌中。相较于应用更为广泛的II型CRISPR系统，I型CRISPR系统的差异在于其需借助多效应因子CASCADE复合物完成CRISPR RNA（crRNA）的加工与靶标识别，同时其标志性核酸酶Cas3具备过程性酶活特性。鉴于I型CRISPR系统分布广泛、Cas3具有过程性酶活特性，且Cas3可产生重组活性单链突出末端，我们推测Cas3可在链霉菌中实现高效基因组工程编辑，具备独特的应用优势。本研究报道了一款基于I型CRISPR系统的链霉菌基因组工程编辑新工具。该质粒系统命名为pCRISPR-Cas3，采用小型化的I-C型CRISPR系统，可实现高效基因组工程编辑。pCRISPR-Cas3的编辑效率优于pCRISPR-Cas9，可介导靶向性缺失及任意长度的基因缺失。此外，本研究证实该工具可有效完成大片段基因组区域的替换，例如生物合成基因簇（biosynthetic gene clusters, BGCs）。无需额外修饰，pCRISPR-Cas3即可在多种链霉菌属物种中实现高效基因组工程编辑。