DNB-based on-chip motif finding: A high-throughput method to profile different types of protein-DNA interactions

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.

PAMPHLET: A Robust Toolkit for Precise PAM Prediction and Unveiling PAM Consistency in Highly Co-occurrence CRISPR-Cas Systems

“…The concentration of DNBs was quantified using the Qubit ssDNA Assay Kit (Thermo Fisher Scientific). PAM identification was carried out according to the method described in Li et al, 2020, with a minor modification: 100 µL of DNBs were loaded onto the BGISEQ-500 V3.1 chip using 33 µL of BGISEQ-500 DNB Loader (MGI Tech Co. Ltd.). Single-end sequencing runs for 43 bases were conducted following the BGISEQ-500RS High-throughput Sequencing Kit instructions.…”

“…Determining functional PAM sequences is a rate-limiting step in the characterization of novel CRISPR- Cas families, as traditional in vivo PAM screening experiments are time-consuming (Tang & Gu, 2020). Despite the availability of high-throughput in vitro PAM determination tools like DocMF based on BGISEQ-500 (Li et al, 2020), the effector proteins and crRNA must be prepared in advance. Thus, a bioinformatics-based PAM prediction method is desirable to streamline the PAM screening process and expedite the characterization of new CRISPR-Cas systems.…”

Exploring molecular biology in sequence space: The road to next-generation single-molecule biophysics

“…Although the biophysical assays are dependent on the amplification chemistry and the immobilization process of the sequencer, they are not bound to a single supplier. The same approach was used with BGI's DNA nanoball sequencing (Li et al, 2020) and could, in principle, be applied to Thermo Fisher's SOLiD sequencing (discontinued), Roche's 454 pyrosequencing (discontinued), and Qiagen's GeneReader platforms (see Box 1). First, the DNA library is sequenced on a sequencing flow cell.…”

“…In the past decade, binding assays on next-generation sequencing chips have been applied to study the influence of DNA, RNA, peptide, and protein sequence for numerous biological systems (Andreasson et al, 2022(Andreasson et al, , 2020Becker et al, 2019aBecker et al, , 2019bBonilla et al, 2021;Boyle et al, 2017;Buenrostro et al, 2014;Denny and Greenleaf, 2019;Denny et al, 2018;Drees and Fischer, 2021;Jarmoskaite et al, 2019;Jones et al, 2021;Jung et al, 2017;Layton et al, 2019;Li et al, 2020;Mamet et al, 2019;Nutiu et al, 2011;Ober-Reynolds et al, 2022;Ozer et al, 2015;Perkel, 2018;She et al, 2017;Svensen et al, 2016;Tome et al, 2014;Wu et al, 2019Yesselman et al, 2019). In the next decade, we expect to obtain a more detailed picture by transitioning from averaging over the roughly thousand molecules in a cluster to measurements of individual molecules.…”