Description: DNA-protein interactions play pivotal roles in many cellular activities, such as replication, development, and DNA repair. Here we report a DNB-based on-chip Motif Finding (DocMF) system that utilizes next-generation-sequencing chips to profile protein binding or cleaving activity toward several hundred millions or billions of DNA sequences. Using DocMF, we successfully identified a variety of endonuclease recognition sites and the protospacer-adjacent motif (PAM) sequences of different CRISPR effector proteins. Compared with the widely used PAM depletion assay, our DocMF platform can simultaneously screen both 5’ and 3’ sequences adjacent to the protospacer to identify PAMs with high coverage using the same DNB library pool. For the well-studied SpCas9, our DocMF platform not only identified its common PAMs, 5’-NGG-3’, but also revealed a small proportion (~5%) of noncanonical 5’-NAG-3’ PAMs. 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 revealed that VeCas9 requires the PAM sequences 5’-NNNRR-3’, whereas BvCpf1 uses the T-rich PAMs 5’-YYN-3’ for target DNA recognition. Moreover, after slightly changing the experimental protocol and analysis threshold, we observed that dCas9, a DNA binding protein lacking endonuclease activity, preferably binds 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 site preferences of different DNA-binding proteins.

Data type: Raw sequence reads

Sample scope: Synthetic

Relevance: Other

Release date: 2019-11-01

Last updated: 2019-10-11

Data size: 95.07GB