Biology subjects

Begemann, M. B.

Publications and source records attributed to Begemann, M. B..

4 recordsLinked to original sources

bioRxiv2026

Comparative characterization of Cas12a2 orthologs identifies high-activity nucleases for programmable cell elimination

Cas12a2 CRISPR nucleases, including SuCas12a2, have been shown to have extensive collateral activity towards RNA, ssDNA, and dsDNA. This collateral activity results in targeted cell elimination and has applications across biotechnology, agriculture, and human health. We explored the natural genetic diversity of Cas12a2 nucleases and characterized nine novel orthologs in a DNA damage kinetic assay in E. coli. Three new Cas12a2 orthologs (RsCas12a2, SdCas12a2, and HmCas12a2) were shown to have high collateral activity towards DNA. These nucleases are highly divergent from SuCas12a2, have conserved core RuvC catalytic residues, and have sequence diversity in the previously reported aromatic clamp residues required for nucleic acid positioning in the active site. We defined PFS preferences and mismatch tolerance for each high-activity Cas12a2 nuclease, expanding the available Cas12a2 toolbox, and discovered functional differences with obvious impacts on downstream applications.

molecular biology↗

bioRxiv2022

Cas12a2 elicits abortive infection via RNA-triggered destruction of double-stranded DNA

Bacterial abortive infection systems limit the spread of foreign invaders by shutting down or killing infected cells before the invaders can replicate1, 2. Several RNA-targeting CRISPR-Cas systems (e.g., types III and VI) cause Abi phenotypes by activating indiscriminate RNases3-5. However, a CRISPR-mediated abortive mechanism that relies on indiscriminate DNase activity has yet to be observed. Here we report that RNA targeting by the type V Cas12a2 nuclease drives abortive infection through non-specific cleavage of double-stranded (ds)DNA. Upon recognition of an RNA target with an activating protospacer-flanking sequence, Cas12a2 efficiently degrades single-stranded (ss)RNA, ssDNA, and dsDNA. Within cells, the dsDNase activity induces an SOS response and impairs growth, stemming the infection. Finally, we harnessed the collateral activity of Cas12a2 for direct RNA detection, demonstrating that Cas12a2 can be repurposed as an RNA-guided, RNA-targeting tool. These findings expand the known defensive capabilities of CRISPR-Cas systems and create additional opportunities for CRISPR technologies.

microbiology↗

bioRxiv2017

Characterization and Validation of a Novel Group of Type V, Class 2 Nucleases for in vivo Genome Editing

CRISPR-based genome editing is an enabling technology with potential to dramatically transform multiple industries. Identification of additional editing tools will be imperative for broad adoption and application of this technology. A novel Type V, Class 2 CRISPR nuclease system was identified from Microgenomates and Smithella bacterial species (CRISPR from Microgenomates and Smithella, Cms1). This system was shown to efficiently generate indel mutations in the major crop plant rice (Oryza sativa). Cms1 are distinct from other Type V nucleases, are smaller than most other CRISPR nucleases, do not require a tracrRNA, and have an AT-rich protospacer-adjacent motif site requirement. A total of four novel Cms1 nucleases across multiple bacterial species were shown to be functional in a eukaryotic system. This is a major expansion of the Type V CRISPR effector protein toolbox and increases the diversity of options available to researchers.

molecular biology↗

bioRxiv2017

Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucleases

Introduction Paragraph Introduction Paragraph Main Body Online Methods References Precise genome editing of plants has the potential to reshape global agriculture through the targeted engineering of endogenous pathways or the introduction of new traits. To develop a CRISPR nuclease-based platform that would enable higher efficiencies of precise gene insertion or replacement, we screened the Cpf1 nucleases from Francisella novicida and Lachnospiraceae bacterium ND2006 for their capacity to induce targeted gene insertions via homology directed repair. Both nucleases, in the presence of guide RNA and repairing DNA template, were demonstrated to generate precise gene insertions as well as indel mutations at the target site in the rice genome. The frequency of targeted insertions for these Cpf1 nucleases, up to 8%, is higher than most other genome editi ...

plant biology↗