Mechanism of expanded DNA recognition in xCas9

Joint Public Review:

Summary:

Hossain and coworkers investigate the mechanisms of recognition of xCas9, a variant of Cas9 with expanded targeting capability for DNA. They do so by using molecular simulations and combining different flavors of simulation techniques, ranging from long classical MD simulations, to enhanced sampling, to free energy calculations of affinity differences. Through this, the authors are able to develop a consistent model of expanded recognition based on the enhanced flexibility of the protein receptor.

Strengths:

The paper is solidly based on the ability of the authors to master molecular simulations of highly complex systems. In my opinion, this paper shows no major weaknesses. The simulations are carried out in a technically sound way. Comparative analyses of different systems provide valuable insights, even within the well-known limitations of MD. Plus, the authors further investigate why xCas9 exhibits improved recognition of the TGG PAM sequence compared to SpCas9 via well-tempered metadynamics simulations focusing on the binding of R1335 to the G3 nucleobase and the DNA backbone in both SpCas9 and xCas9. In this context, the authors provide a free-energy profiling that helps support their final model.

The implementation of FEP calculations to mimic directed evolution improvement of DNA binding is also interesting, original and well-conducted.

Overall, my assessment of this paper is that it represents a strong manuscript, competently designed and conducted, and highly valuable from a technical point of view.

Weaknesses:

To make their impact even more general, the authors may consider expanding their discussion on entropic binding to other recent cases that have been presented in the literature recently (such as e.g. the identification of small molecules for Abeta peptides, or the identification of "fuzzy" mechanisms of binding to protein HMGB1). The point on flexibility helping adaptability and expansion of functional properties is important, and should probably be given more evidence and more direct links with a wider picture.