CRISPR-based transcriptional regulation technologies, including CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi), offer precise and programmable control over gene expression, representing a major advance in gene and epigenetic therapy. CRISPRa uses nuclease-inactive Cas proteins fused to transcriptional activators to upregulate target genes, while CRISPRi employs repressor domains for gene silencing. Preclinical studies have demonstrated the efficacy of CRISPRa/i in models of metabolic, neurological, muscular, and oncological diseases. Notably, CRISPRi-based therapies have entered clinical trials for conditions like hepatitis B and muscular dystrophy, showing encouraging safety and efficacy profiles. Despite ongoing challenges related to delivery efficiency, immunogenicity, and off-target activity, innovations in protein engineering and guide RNA design are rapidly enhancing the precision and safety of these technologies. Overall, CRISPRa and CRISPRi are poised to transform the treatment of genetic and epigenetic disorders, with continued optimization expected to accelerate their clinical adoption and broaden their therapeutic impact.
In this paper we present a theoretical design for tile-based self-assembling systems where individual tile sets that are universal for various tasks (e.g. building shapes or encoding arbitrary data for algorithmic systems) can be provided their input solely using sequences of variations in temperatures. Although there is prior theoretical work in so-called "temperature programming," the results presented here are based upon recent experimental work with "blocked tiles" that provides plausible evidence for their practical implementation. We develop and present an abstract mathematical model, the BlockTAM, for such systems that is based upon the experimental work, and provide constructions within it for universal number encoding systems and a universal shape building construction. These results mirror previous results in temperature programming, covering the two ends of the spectrum that seek to balance the scale factors of assemblies with the number of temperature phases required, while leveraging the features of the BlockTAM that we hope provide a pathway for future experimental implementations.
For general discrete Chemical Reaction Networks (CRNs), the fundamental problem of reachability - the question of whether a target configuration can be produced from a given initial configuration - was recently shown to be Ackermann-complete. However, many open questions remain about which features of the CRN model drive this complexity. We study a restricted class of CRNs with void rules, reactions that only decrease species counts. We further examine this regime in the motivated model of step CRNs, which allow additional species to be introduced in discrete stages. With and without steps, we characterize the complexity of the reachability problem for CRNs with void rules. We show that, without steps, reachability remains polynomial-time solvable for bimolecular systems but becomes NP-complete for larger reactions. Conversely, with just a single step, reachability becomes NP-complete even for bimolecular systems. Our results provide a nearly complete classification of void-rule reachability problems into tractable and intractable cases, with only a single exception.
DNA strand displacement, a collective name for certain behaviors of short strands of DNA, has been used to build many interesting molecular devices over the past few decades. Among those devices are general implementation schemes for Chemical Reaction Networks, suggesting a place in an abstraction hierarchy for complex molecular programming. However, the possibilities of DNA strand displacement are far from fully explored. On a theoretical level, most DNA strand displacement systems are built out of a few simple motifs, with the space of possible motifs otherwise unexplored. On a practical level, the desire for general, large-scale DNA strand displacement systems is not fulfilled. Those systems that are scalable are not general, and those that are general don't scale up well. We have recently been exploring the space of possibilities for DNA strand displacement systems where all input complexes are made out of at most two strands of DNA. As a test case, we've had an open question of whether such systems can implement general Chemical Reaction Networks, in a way that has a certain set of other desirable properties - reversible, systematic, O(1) toeholds, bimolecular reactions, and correct according to CRN bisimulation - that the state-of-the-art implementations with more than 2-stranded inputs have. Until now we've had a few results that have all but one of those desirable properties, including one based on a novel mechanism we called coupled reconfiguration, but that depended on the physically questionable assumption that pseudoknots cannot occur. We wondered whether the same type of mechanism could be done in a pseudoknot-robust way. In this work we show that in fact, coupled reconfiguration can be done in a pseudoknot-robust way, and this mechanism can implement general Chemical Reaction Networks with all inputs being single strands of DNA. Going further, the same motifs used in this mechanism can implement stacks and surface-based bimolecular reactions. Those have been previously studied as part of polymer extensions of the Chemical Reaction Network model, and on an abstract model level, the resulting extensions are Turing-complete in ways the base Chemical Reaction Network model is not. Our mechanisms are significantly different from previously tested DNA strand displacement systems, which raises questions about their ability to be implemented experimentally, but we have some reasons to believe the challenges are solvable. So we present the pseudoknot-robust coupled reconfiguration mechanism and its use for general Chemical Reaction Network implementations; we present the extensions of the mechanism to stack and surface reactions; and we discuss the possible obstacles and solutions to experimental implementation, as well as the theoretical implications of this mechanism.
A grand challenge facing molecular programmers is the rational development of fast, robust, and isothermal architectures akin to "chemical central processing units" that can sense (bio-)chemical signals from their environment, perform complex computation, and orchestrate a physical response in situ. DNA strand displacement systems (DSDs) remain a compelling candidate, but are hampered by spurious reaction pathways that lead to incorrect output. DSDs that utilize the systematic leakless motif can be made arbitrarily robust at the cost of increasing redundancy and network size (scaling), and thus a degradation of kinetic performance. Another class of architectures utilize DNA hybridization, extension, and signal production of entirely sequestered outputs via strand-displacing polymerases (SDPs) that have resulted in impressive demonstrations; however, they face similar challenges of aberrant behavior such as mis-priming by incorrect signals. Our work introduces a unified polymerase-dependent toehold-mediated strand displacement (PD-TMSD) architecture that integrates the programmed specificity of DSDs with the unique advantages of SDPs. This unification enables systems that can be made arbitrarily robust, at any concentration range, without increasing network size. We propose a number of gate designs and composition rules to compute arbitrary Boolean functions, emulate arbitrary chemical reaction networks, and explore time-bounded probabilistic computation made possible by certain classes of SDPs. Our theoretical exploration is backed by preliminary experimental demonstrations. This contribution was inspired by the belief that molecular programming can meet or exceed the complexity exhibited in biology if we embrace its best understood molecular machinery and couple it with systematic design principles built upon a strong theoretical foundation.
