More Like this

1. DNA Cleavage by a De Novo Designed Protein–Titanium Complex

   https://doi.org/10.1021/acs.inorgchem.0c01707  

   Paredes, Alexander; Loh, Brittany M.; Peduzzi, Olivia M.; Reig, Amanda J.; Buettner, Katherine M. (August 2020, Inorganic Chemistry)

   null (Ed.)
2. Enhancement of CRISPR/Cas12a trans -cleavage activity using hairpin DNA reporters

   https://doi.org/10.1093/nar/gkac578  

   Rossetti, Marianna; Merlo, Rosa; Bagheri, Neda; Moscone, Danila; Valenti, Anna; Saha, Aakash; Arantes, Pablo R; Ippodrino, Rudy; Ricci, Francesco; Treglia, Ida; et al (July 2022, Nucleic Acids Research)

   Abstract 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. 

   more » « less
3. Catalytic Mechanism of Non-Target DNA Cleavage in CRISPR-Cas9 Revealed by Ab Initio Molecular Dynamics

   https://doi.org/10.1021/acscatal.0c03566  

   Casalino, Lorenzo; Nierzwicki, Łukasz; Jinek, Martin; Palermo, Giulia (November 2020, ACS Catalysis)

   null (Ed.)
4. Assessing Temperature-Dependent DNA Cleavage by CRISPR-Cas9

   https://doi.org/10.21769/BioProtoc.5463  

   Knight, Alexa; Luo, Jinping; Lisi, George (January 2025, BIO-PROTOCOL)
5. Lentiviral CRISPRa/i in the adult prairie vole brain: modulating neuronal gene expression without DNA cleavage

   https://doi.org/10.3389/fgeed.2025.1602983  

   Loth, Meredith K; Mesch, Kendall T; Herrera-Garcia, Celine; Brusman, Liza E; Donaldson, Zoe R (May 2025, Frontiers in Genome Editing)

   Prairie voles (Microtus ochrogaster) are a powerful model for studying the neurobiology of social bonding, yet tools for region- and cell type-specific gene regulation remain underdeveloped in this species. Here, we present a lentivirus-mediated CRISPR activation and interference (CRISPRa/i) platform for somatic gene modulation in the prairie vole brain. This system enables non-mutagenic, titratable regulation of gene expression in the adult brain without germline modification. Our dual-vector system includes one construct expressing dCas9-VPR (VP64-p65-Rta) referred to as CRISPRa or dCas9-KRAB-MeCP2 (Kruppel-associated box-methyl CpG binding protein 2), referred to as CRISPRi under a neuron-specific promoter, and a second construct delivering a U6-driven sgRNA (single guide RNA) alongside an elongation factor 1 alpha (EF1α)-driven mCherry reporter. We detail the design, production, and stereotaxic delivery of these tools and demonstrate their application by targeting four genes implicated in social behavior (Oxtr, Avpr1a, Drd1,andDrd2) across two mesolimbic brain regions: the nucleus accumbens and ventral pallidum. Gene expression analyses confirmed robust, bidirectional transcriptional modulation for selected targets, establishing a proof of concept for CRISPRa/i in this non-traditional model. The dual-vector design is readily adaptable to other gene targets, cell types, and brain regions, and can be multiplexed to provide a flexible and scalable framework for investigating gene function in behaviorally relevant circuits. These advances represent the first successful implementation of somatic CRISPRa/i in prairie voles and expand the genetic toolkit available for this species. 

   more » « less