DNB-based on-chip motif finding: A high-throughput method to profile different types of protein-DNA interactions
Zhuokun Li, Xiaojue Wang, Dongyang Xu, Dengwei Zhang, Dan Wang, Xuechen Dai, Qi Wang, Zhou Li, Ying Gu, Wenjie Ouyang, Shuchang Zhao, Baoqian Huang, Jian Gong, Jing Zhao, Ao Chen, Yue Shen, Yuliang Dong, Wenwei Zhang, Xun Xu, Chongjun Xu, Yuan Jiang
Abstract
Here, we report a sensitive DocMF system that uses next-generation sequencing chips to profile protein-DNA interactions. Using DocMF, we successfully identified a variety of endonuclease recognition sites and the protospacer adjacent motif (PAM) sequences of different CRISPR systems. DocMF can simultaneously screen both 5' and 3' PAMs with high coverage. For SpCas9, we found noncanonical 5'-NAG-3' (~5%) and 5'-NGA-3' (~1.6%), in addition to its common PAMs, 5'-NGG-3' (~89.9%). More relaxed PAM sequences of two uncharacterized Cas endonucleases, VeCas9 and BvCas12a, were extensively characterized using DocMF. Moreover, we observed that dCas9, a DNA binding protein lacking endonuclease activity, preferably bound to the previously reported 5'-NGG-3' sequence. In summary, our studies demonstrate that DocMF is the first tool with the capacity to exhaustively assay both the binding and the cutting properties of different DNA binding proteins.