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CRISPR-based DNA adenine base editors (ABEs) hold remarkable promises to address human genetic diseases caused by point mutations. ABEs were developed by combining CRISPR-Cas9 with a transfer RNA (tRNA) adenosine deaminase enzyme and through directed evolution, conferring the ability to deaminate DNA. However, the molecular mechanisms driving the efficient DNA deamination in the evolved ABEs remain unresolved. Here, extensive molecular simulations and biochemical experiments reveal the biophysical basis behind the astonishing base editing efficiency of ABE8e, the most efficient ABE to date. We demonstrate that the ABE8e’s DNA deaminase domain, TadA8e, forms remarkably stable dimers compared to its tRNA-deaminating progenitor and that the strength of TadA dimerization is crucial for DNA deamination. The TadA8e dimer forms robust interactions involving its R98 and R129 residues, the RuvC domain of Cas9 and the DNA. These locking interactions are exclusive to ABE8e, distinguishing it from its predecessor, ABE7.10, and are indispensable to boost DNA deamination. Additionally, we identify three critical residues that drive the evolution of ABE8e toward improved base editing by balancing the enzyme’s activity and stability, reinforcing the TadA8e dimer and improving the ABE8e’s functionality. These insights offer new directions to engineer superior ABEs, advancing the design of safer precision genome editing tools.

 

CRISPR-Cas12a is a powerful RNA-guided genome-editing system that generates double-strand DNA breaks using its single RuvC nuclease domain by a sequential mechanism in which initial cleavage of the non-target strand is followed by target strand cleavage. How the spatially distant DNA target strand traverses toward the RuvC catalytic core is presently not understood. Here, continuous tens of microsecond-long molecular dynamics and free-energy simulations reveal that an α-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the DNA target strand by anchoring the crRNA:target strand duplex and guiding the target strand toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the REC2 domain pushes the crRNA:target strand duplex toward the core of the enzyme, while the Nuc domain aids the bending and accommodation of the target strand within the RuvC core by bending inward. Understanding of this critical process underlying Cas12a activity will enrich fundamental knowledge and facilitate further engineering strategies for genome editing.

 

Metadynamics calculations of large chemical systems with ab initio methods are computationally prohibitive due to the extensive sampling required to simulate the large degrees of freedom in these systems. To address this computational bottleneck, we utilized a GPU-enhanced density functional tight binding (DFTB) approach on a massively parallelized cloud computing platform to efficiently calculate the thermodynamics and metadynamics of biochemical systems. To first validate our approach, we calculated the free-energy surfaces of alanine dipeptide and showed that our GPU-enhanced DFTB calculations qualitatively agree with computationally-intensive hybrid DFT benchmarks, whereas classical force fields give significant errors. Most importantly, we show that our GPU-accelerated DFTB calculations are significantly faster than previous approaches by up to two orders of magnitude. To further extend our GPU-enhanced DFTB approach, we also carried out a 10 ns metadynamics simulation of remdesivir, which is prohibitively out of reach for routine DFT-based metadynamics calculations. We find that the free-energy surfaces of remdesivir obtained from DFTB and classical force fields differ significantly, where the latter overestimates the internal energy contribution of high free-energy states. Taken together, our benchmark tests, analyses, and extensions to large biochemical systems highlight the use of GPU-enhanced DFTB simulations for efficiently predicting the free-energy surfaces/thermodynamics of large biochemical systems. 

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.