Biology subjects

Lukarska, M.

Publications and source records attributed to Lukarska, M..

5 recordsLinked to original sources

bioRxiv2026

Activity and specificity trade-offs in adenine base editors

Adenine base editors (ABEs) are CRISPR effectors that introduce A-T to G-C transitions in the genome using a nucleotide deaminase fused to a Cas protein. ABEs have been evolved to have very high editing efficiency, but off-target editing effects compromise their precision and hinder their applications. Here we explore the activity and specificity relationship of ABEs using a combination of machine learning-guided design and high-throughput screening. We designed a diverse library of 12,000 variants and built quantitative bacterial selection systems that allowed us to simultaneously measure their on-target and off-target editing. We found that the ABEs were fully described by the single dimension of intrinsic deaminase activity with no evidence for independent specialization with respect to local sequence context, editing window width, RNA editing, or genotoxicity. These results were supported by in vitro studies and consistent with editing experiments in mammalian cells. Finally, the activity and specificity trade-offs were recapitulated among previously reported engineered variants and a selection of library variants spanning the activity spectrum. Our results suggest that fundamental architectural improvements will be necessary to transcend the activity and specificity limitations for the next generation of ABEs.

molecular biology↗

bioRxiv2025

Exploring the deletion landscape of S. aureus Cas9 with SABER

Profiling tolerated amino acid deletions in proteins can elucidate structure-function relationships, reconstruct intermediate stages in protein evolution, and be used to engineer minimized versions of proteins with size-sensitive biotechnology applications. Despite advances in deletion library construction techniques over the past several decades, there are presently few methods available that are simultaneously efficient, precise, and easy to implement. Here we present SABER, a novel approach which utilizes SpRYCas9, a near-PAMless engineered SpCas9 variant, as a molecular biological tool for building deletion libraries with unprecedented speed and ease. We applied this technique to the small and structurally divergent Cas9 from Staphylococcus aureus (SaCas9) and mapped the set of deletions tolerated for DNA binding activity. We proceeded to use this information to design a set of minimal SaCas9-based effectors capable of CRISPRi transcriptional repression in bacterial cells. Our findings provide new insights into the function of certain structural elements in SaCas9, and we anticipate that our dSaCas9 deletion map may prove useful in further efforts to develop minimal Cas9-based effectors and gene editors.

molecular biology↗

bioRxiv2024

Mechanism of Co-Transcriptional Cap-Snatching by Influenza Polymerase

Influenza virus mRNA is stable and competent for nuclear export and translation because it receives a 5' cap(1) structure in a process called cap-snatching1. During cap-snatching, the viral RNA-dependent RNA polymerase (FluPol) binds to host RNA polymerase II (Pol II) and the emerging transcript2,3. The FluPol endonuclease then cleaves a capped RNA fragment that sub-sequently acts as a primer for the transcription of viral genes4,5. Here, we present the cryo-EM structure of FluPol bound to a transcribing Pol II in complex with the elongation factor DSIF in the pre-cleavage state. The structure shows that FluPol directly interacts with both Pol II and DSIF, which position the FluPol endonuclease domain near the RNA exit channel of Pol II. These interactions are important for the endonuclease activity of FluPol and FluPol activity in cells. A second structure trapped after cap-snatching shows that cleavage rearranges the capped RNA primer within the FluPol, directing the capped RNA 3'-end towards the FluPol polymerase active site for viral transcription initiation. Altogether, our results provide the molecular mechanisms of co-transcriptional cap-snatching by FluPol.

biochemistry↗

bioRxiv2022

Type B and Type A influenza polymerases have evolved distinct binding interfaces to recruit the RNA polymerase II CTD

During annual influenza epidemics, influenza B viruses (IBVs) co-circulate with influenza A viruses (IAVs), can become predominant and cause severe morbidity and mortality. Phylogenetic analyses suggest that IAVs (primarily avian viruses) and IBVs (primarily human viruses) have diverged over long time scales. Identifying their common and distinctive features is an effective approach to increase knowledge about the molecular details of influenza infection. The virus-encoded RNA-dependent RNA polymerases (FluPolB and FluPolA) are PB1-PB2-PA heterotrimers that perform transcription and replication of the viral genome in the nucleus of infected cells. Initiation of viral mRNA synthesis requires a direct association of FluPol with the host RNA polymerase II (RNAP II), in particular the repetitive C-terminal domain (CTD) of the major RNAP II subunit, to enable "cap-snatching" whereby 5-capped oligomers derived from nascent RNAP II transcripts are pirated to prime viral transcription. Here, we present the first high-resolution co-crystal structure of FluPolB bound to a CTD mimicking peptide at a binding site crossing from PA to PB2. By performing structure-based mutagenesis of FluPolB and FluPolA followed by a systematic investigation of FluPol-CTD binding, FluPol activity and viral phenotype, we demonstrate that IBVs and IAVs have evolved distinct binding interfaces to recruit the RNAP II CTD, despite the CTD sequence being highly conserved across host species. We find that the PB2 627 subdomain, a major determinant of FluPol-host cell interactions and IAV host-range, is involved in CTD-binding for IBVs but not for IAVs, and we show that FluPolB and FluPolA bind to the host RNAP II independently of the CTD. Altogether, our results strongly suggest that the CTD-binding modes of IAV and IBV represent avian- and human-optimized binding modes, respectively, and that their divergent evolution was shaped by the broader interaction network between the FluPol and the host transcriptional machinery. Authors summaryDuring seasonal influenza epidemics, influenza B viruses (IBVs) co-circulate with influenza A viruses (IAVs) and can cause severe outcomes. The influenza polymerase is a key drug target and it is therefore important to understand the common and distinctive molecular features of IBV and IAV polymerases. To achieve efficient transcription and replication in the nucleus of infected cells, influenza polymerases closely cooperate with the cellular RNA polymerase II (RNAP II) and interact with the repetitive C-terminal domain (CTD) of its major subunit. Here we gained new insights into the way IBV and IAV polymerases interact with the CTD of RNAP II. High-resolution structural data was used to perform structure-based mutagenesis of IBV and IAV polymerases followed by a systematic investigation of their interaction with RNAP II, transcription/replication activity and viral phenotype. Strikingly, we found that IBVs and IAVs have evolved distinct interfaces to interact with the host transcriptional machinery, in particular with the CTD of RNAP II. We provide evidence that these differences may have evolved as a consequence of the differences in IBV and IAV host range. Our findings are of significant importance with regard to the development of broad-spectrum antivirals that target the virus-host interface.

microbiology↗

bioRxiv2020

Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules

Proteins evolve through the modular rearrangement of elements known as domains. It is hypothesized that extant, multidomain proteins are the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) that comprehensively assays all possible deletions of a protein. Using MISER, we generated a deletion landscape for the CRISPR protein Cas9. We found that Cas9 can tolerate large single deletions to the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.

molecular biology↗