CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection against intruding genomic materials in bacteria and archaea. The complementarity of CRISPR RNA (crRNA) guides Cas protein to a target DNA and/or RNA, which then initiates strand cleavage by a specific Cas protein. Cas9, a type II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA (sgRNA) can direct Cas9 to cleave DNA targets of interest. In addition to the complementarity between the guide region of the sgRNA and the target DNA, a three to eight nucleotide long region, termed protospacer adjacent motif (PAM), is essential for Cas9 function. The relationship between RNA-mediated conformational changes in Cas9 and DNA targeting is being pursued actively for developing Cas9 variants with high specificity for gene editing. Our lab has focused our efforts on identifying how an arginine-rich bridge helix (BH) present in Cas9 contributes conformational checkpoints essential for stringent DNA cleavage (1). Cas9 offers great promises for gene therapy applications and one critical aspect for such applications is to completely understand the DNA cleavage requirements of Cas9 and identify any promiscuous DNA cleavage by this protein. Interestingly, our research has identified conditions where Cas9 and other Cas proteins can cleave DNA in the absence of a guide RNA, under in vitro conditions (2). Such promiscuous activities can be detrimental if conditions that can trigger RNA-independent DNA cleavage exist under gene therapy or gene editing conditions. Another aspect of our research is to understand how bacteria get immunized against phage infections. We analyse the protein-nucleic acid interactions that are essential for site-specific insertion of foreign DNA into the bacterial CRISPR locus to provide immunity against future infections (3). Together, our studies develop mechanistic insights into CRISPR-Cas systems that are valuable in developing Cas9 as an error-proof gene editing system as well as to develop novel CRISPR-Cas tools for other biotech applications such as gene tagging.

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One of the fundamental questions common to all of the neurodegenerative diseases is why a particular disease targets specific neuronal populations? Excitatory neurons in layer II of the entorhinal cortex (EC) and the CA1 region of the hippocampus are preferentially impaired in early Alzheimer's disease (AD), but the pathways contributing to their relative vulnerability remain largely unknown. Pathological tau, a major pathological hallmark of AD predominantly accumulates in certain excitatory neurons, but not inhibitory neurons or glial cells in those brain regions affected in early AD. By analyzing RNA transcripts from single-nucleus RNA datasets, we identified a specific tau homeostasis signature of genes differentially expressed in excitatory compared to inhibitory neurons. One of the genes, BCL2-associated athanogene 3 (BAG3), a facilitator of autophagy, was identified as a hub, or master regulator, gene. We verified that reducing BAG3 levels in primary neurons exacerbated pathological tau accumulation, whereas BAG3 overexpression attenuated it. These results define a tau homeostasis signature that underlies the cellular and regional vulnerability of excitatory neurons to tau pathology. Our ongoing research focuses on understanding which subtypes of excitatory neurons are vulnerable to tau pathology in early AD and the molecular and cellular mechanisms underlying the selective neuronal vulnerability. We are also very interested in investigating the role of non-autonomous (microglia, astrocytes and/or oligodendrocytes) effects and aging in selective neuronal vulnerability to tauopathies in AD.