Engineered DNA-binding proteins that manipulate the human genome and 
transcriptome have enabled 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 capability 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 induction. 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 polymerase III promoters that are incorporated into 
the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently 
expressed and can mediate multiplex gene editing and sustained transcriptional 
activation in immortalized and primary human cells. This delivery system will be 
significant to enabling the potential of CRISPR/Cas9-based multiplex genome 
engineering in diverse cell types.