Nuclear localization signal (NLS) is a short peptide guiding the nuclear transport process, recognized as playing an important role in constructing clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) activators. Here, we investigate the effect of the position and number of the NLS on transcriptional activation based on the dCas9-VPR activator. Our results not only demonstrate that the position of the SV40 NLS could have different degrees of influence on activation efficiency but also, surprisingly, we find that the SV40 NLS plays a detrimental role. Complete deletion of the NLS from the system could increase the transcriptional activation efficiency by 2 to 4 times compared with the original dCas9-VPR. This finding is also supported by some typical first- and third-generation activators. Our work should be beneficial to the design of the NLS-based system.