Nuclease-deactivated Cas9 fused to the VP64 activation domain (dCas9-VP64)
CRISPR activator fusing nuclease-null Cas9 to VP64, four tandem copies of the herpes simplex virus VP16 activation domain. A guide RNA directs it to a promoter, where it raises expression of the endogenous gene without cutting DNA. Single guide RNAs activate only modestly, so several per promoter are used to act synergistically.
Length: 4395 bp (1465 aa)
Function: activation
Base protein: dCas9 Catalytically Dead Nuclease
Origin: dCas9 fused to the VP64 activation domain
Characteristics
VP64 is fused directly to the C terminus of nuclease-null Cas9, which binds the guide RNA target without cutting it. The fusion activates reporter constructs robustly with guide RNAs targeting sequences near the promoter. At endogenous genes, individual guide RNAs stimulate transcription only modestly, while several acting together give robust multi-fold induction. Guide RNAs closer to the transcription start site contribute most of the stimulation, and guide RNAs within about 1 kb upstream activated SOX2 and NANOG robustly. An alternative two-component form recruits VP64 to the guide RNA instead, through two MS2 stem-loops on the guide RNA 3' end and an MS2-VP64 fusion.
Applications: Activation of endogenous human genes with several guide RNAs per promoter, shown for REX1 with 10 guide RNAs across 5 kb upstream of the transcription start site, and for SOX2 and NANOG within 1 kb upstream. Used as the reference activator that stronger systems are measured against, including dCas9-VPR and dCas9-SunTag-VP64.
Limitations: A single guide RNA gives weak activation, so robust activation needs several guide RNAs per promoter or a stronger system such as dCas9-VPR or dCas9-SunTag-VP64. dCas9 derives from SpCas9, so targets need an NGG PAM, and its roughly 4.1 kb coding sequence leaves little room for other elements in a single AAV vector.
Architecture: Nuclease-null Cas9 fused at its C terminus to VP64, a tetrameric repeat of the VP16 minimal activation domain from herpes simplex virus.
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