Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector

Engineered DNA-binding proteins that manipulate the human genome and transcriptome have en-abled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capabil-ity of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induc-tion. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA poly-merase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can medi-ate multiplex gene editing and sustained transcrip-tional activation in immortalized and primary human cells. This delivery system will be significant to en-abling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types