Rxivist logo

DNB-Based On-Chip Motif Finding (DocMF): a High-Throughput Method to Profile Different Types of Protein-DNA Interactions

By 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

Posted 01 Nov 2019
bioRxiv DOI: 10.1101/827428 (published DOI: 10.1126/sciadv.abb3350)

Here we report a highly sensitive DNB-based on-chip Motif Finding (DocMF) system that utilizes high throughput next-generation-sequencing (NGS) chips to profile protein binding or cleaving activity. Using DocMF, we successfully identified a variety of endonuclease recognition sites and the protospacer-adjacent-motif (PAM) sequences of different CRISPR systems. Our DocMF platform can simultaneously screen both 5’ and 3’ PAM regions with high coverage using the same NGS library/chip. For the well-studied SpCas9, our DocMF platform identified a small proportion of noncanonical 5’-NAG-3’ (∼5%) and 5’-NGA-3’ (∼1.6%), in addition to its common PAMs, 5’-NGG-3’ (∼89.9%). We also used the DocMF to assay two uncharacterized Cas endonucleases, VeCas9 and BvCpf1. VeCas9 PAMs were not detected by the conventional PAM depletion method. However, DocMF discovered that both VeCas9 and BvCpf1 required broader and more complicated PAM sequences for target recognition. VeCas9 preferred the R-rich motifs, whereas BvCpf1 used the T-rich PAMs. Moreover, after slightly changing the experimental protocol, we observed that dCas9, a DNA-binding protein lacking endonuclease activity, preferably binded to the previously reported PAMs 5’-NGG-3’. 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.

Download data

  • Downloaded 435 times
  • Download rankings, all-time:
    • Site-wide: 66,898
    • In molecular biology: 1,910
  • Year to date:
    • Site-wide: 100,794
  • Since beginning of last month:
    • Site-wide: 94,773

Altmetric data


Downloads over time

Distribution of downloads per paper, site-wide


PanLingua

News