CRISPR/Cas technologies have rapidly become in routine use for site-directed genetic or transcriptional manipulation. Despite this, the efficiency of CRISPR/Cas9 functioning cannot entirely be predicted, and it is not fully understood which factors contribute to this variability. Recent studies indicate that heterochromatin can negatively affect Cas9 binding and functioning. Investigating chromatin factors indicates that DNA cytosine-5 methylation does not directly block Cas9 binding. Nucleosomes, however, can completely block Cas9 access to DNA in cell-free assays and present a substantial hurdle in vivo. In addition to being associated with an open chromatin state, active transcription can directly stimulate DNA cleavage by influencing Cas9 release rates in a strand-specific manner. With these insights and a better understanding of genome-wide chromatin and transcription states, CRISPR/Cas9 effectiveness and reliability can be improved.

CRISPR–Cas9-based “gene drive” technologies have been proposed as a novel and effective means of controlling human diseases vectored by mosquitoes. However, more complex designs than those demonstrated to dateand an expanded molecular toolbox with which to build themwill be required to overcome the issues of resistance formation/evolution and drive spatial/temporal limitation. Foreseeing this need, we assessed the sgRNA transcriptional activities of 33 phylogenetically diverse insect Polymerase III promoters using three disease-relevant Culicine mosquito cell lines (Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus). We show that U6 promoters work across species with a range of transcriptional activity levels and find 7SK promoters to be especially promising because of their broad phylogenetic activity. We further show that U6 promoters can be substantially truncated without affecting transcriptional levels. These results will be of great utility to researchers involved in developing the next generation of gene drives.