The RNA-guided endonuclease Cas9 (CRISPR-Cas9) genome editing system has been widely used for biomedical research and holds great potential for therapeutic applications in eukaryotes. The conventional vector-based CRISPR-Cas9 delivery system requires two different RNA polymerase promoters for expression of the guide RNA (gRNA) and Cas9 endonuclease. The large size and relative complexity of such CRISPR transgene cassettes impede their broad implementation, especially in gene therapy applications with viral vectors that have a limited packaging capacity. Here, we report the design of a single-promoter-driven CRISPR-Cas9 system that uses the dual-polymerase (Pol II and Pol III) activity of the H1 promoter. This size reduction strategy of the vector insert provides a significant titer advantage in the lentiviral vector over the regular CRISPR system.

RNA引导的核酸内切酶Cas9（CRISPR-Cas9）基因组编辑系统已广泛用于生物医学研究，并在真核生物中具有广阔的治疗应用前景。基于常规载体的CRISPR-Cas9递送系统需要两个不同的RNA聚合酶启动子来表达指导RNA（gRNA）和Cas9核酸内切酶。这种CRISPR转基因盒的大尺寸和相对复杂性阻碍了它们的广泛实施，特别是在具有有限包装能力的病毒载体的基因治疗应用中。在这里，我们报告了一个由单启动子驱动的CRISPR-Cas9系统的设计，该系统使用了H1启动子的双重聚合酶（Pol II和Pol III）活性。载体插入物的这种减小尺寸的策略在慢病毒载体中比常规CRISPR系统具有明显的滴度优势。