The results of this work will impact the field in several ways. First, the reduced binding efficiency of CRISPR-Cas9 to target DNA with GQ forming sequence suggests that there are limitations about what type of sequences can be targeted by Cas9. Secondly, binding of CRISPR-Cas9 to TS facilitates secondary structure formation in the non-target strand. If these structures are stable enough, they could induce significant distortions in the resulting complex. We have observed significant dynamics in the complex in both of these scenarios, highlighting the underlying competition between secondary structure formation and Watson-Crick pairing. In addition, if NTS can form GQ, the corresponding crRNA could also form GQ. We showed that crRNA loading to Cas9 becomes less efficient if the crRNA contains a GQ forming sequence. These types of issues with crRNA loading to Cas9 might be avoidable by introducing one or two nucleotide mutations in the distal end of crRNA (away from protospacers adjacent motif-PAM). In general, it is possible to eliminate GQ formation by mutating 1-2 guanines. When such a mutation is made, the complementarity of crRNA with the target strand will be lost for the mutated nucleotide(s); however, this might have minimal impact on binding stability and specificity of CRISPR-Cas9 to target dsDNA if a guanine in the distal region is mutated. The impact of such a mutation on Cas9 cleavage activity would require further investigation.
By using a catalytically inactive Cas9 (dCas9), it should be possible to modulate GQ stability without cleaving the DNA. By targeting either the GQ forming G-rich strand or the complementary cytosine-rich (C-rich) strand, it should be possible to inhibit or promote GQ formation, respectively. This is potentially significant as it could be used to target GQ-forming sequences or their complementary C-rich strands in promoters to modulate gene expression. This method would be more specific compared to the alternative method of targeting such sequences with small molecules that have structural but not sequence specificity.
This initial work will be continued in the form of an international collaboration between my lab in USA and Dr. Joo’s lab in Netherlands. The project was expanded from studying one to three GQ forming sequences with different stabilities. This expansion provided a better view about the range of impact that these GQs could have on CRISPR-Cas9 activity. However, certain aspects of the work could not be completed within the 1-year period. The remaining part of the work will be completed by Balci in USA and V. Globyte from the Joo lab in Netherlands. Balci will seek further funding for this work in USA. Finally, Balci was involved with training of undergraduate and graduate students at Delft. In particular, he served as the daily supervisor (along with Mr. Tao Ju Cui) for a master’s student (Rodrigo G. Linares), which served the two-way exchange goal aimed by the Marie Curie Fellowship.
