Despite recent advances in genome editing capabilities for the model organism Saccharomyces cerevisiae, the chromosomal integration of large biochemical pathways for stable industrial production remains challenging. In this work, we developed a simple platform for high-efficiency, single-step, markerless, multi-copy chromosomal integration of full biochemical pathways in Saccharomyces cerevisiae. In this Di-CRISPR (delta integration CRISPR-Cas) platform based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas), we specifically designed guide RNA sequences to target multiple delta sites in the yeast genome. The generation of double stranded breaks at the delta sites allowed simultaneous integration of multiple copies of linearized donor DNA containing large biochemical pathways. With our newly developed Di-CRISPR platform, we were able to attain highly efficient and markerless integration of large biochemical pathways and achieve an unprecedented 18-copy genomic integration of a 24 kb combined xylose utilization and (R,R)-2,3-butanediol (BDO) production pathway in a single step, thus generating a strain that was able to produce BDO directly from xylose. The simplicity and high efficiency of the Di-CRISPR platform could provide a superior alternative to high copy plasmids and would render this platform an invaluable tool for genome editing and metabolic engineering in S. cerevisiae.

尽管最近对于模型生物酿酒酵母的基因组编辑能力有了进步，但是为稳定工业生产而进行的大型生化途径的染色体整合仍然具有挑战性。在这项工作中，我们开发了一个简单的平台，用于酿酒酵母中完整的生化途径的高效，单步，无标记，多拷贝染色体整合。在基于成簇的规则间隔短回文重复序列（CRISPR）和CRISPR相关系统（Cas）的Di-CRISPR（δ整合CRISPR-Cas）平台中，我们专门设计了靶向RNA序列的序列以靶向酵母基因组中的多个δ位点。在三角洲位点的双链断裂的产生允许同时整合包含大的生化途径的线性化供体DNA的多个拷贝。借助我们新开发的Di-CRISPR平台，我们能够实现大型生化途径的高效无标记整合，并实现了空前的18拷贝基因组整合，即24 kb结合木糖利用率和（R，R）-2,3-丁二醇（BDO）的生产过程仅一步之遥，因此产生了能够直接从木糖生产BDO的菌株。Di-CRISPR平台的简单性和高效性可以为高拷贝质粒提供卓越的替代方案，并使该平台成为酿酒酵母基因组编辑和代谢工程的宝贵工具。