Nat Rev Drug Discov
December 2024
Clustered regularly interspaced short palindromic repeats (CRISPR) technology has transformed molecular biology and the future of gene-targeted therapeutics. CRISPR systems comprise a CRISPR-associated (Cas) endonuclease and a guide RNA (gRNA) that can be programmed to guide sequence-specific binding, cleavage, or modification of complementary DNA or RNA. However, the application of CRISPR-based therapeutics is challenged by factors such as molecular size, prokaryotic or phage origins, and an essential gRNA cofactor requirement, which impact efficacy, delivery and safety.
View Article and Find Full Text PDFHuntington's disease (HD) is an incurable neurodegenerative disorder caused by genetic expansion of a CAG repeat sequence in one allele of the () gene. Reducing expression of the mutant HTT (mutHTT) protein has remained a clear therapeutic goal, but reduction of wild-type HTT (wtHTT) is undesirable, as it compromises gene function and potential therapeutic efficacy. One promising allele-selective approach involves targeting the CAG repeat expansion with steric binding small RNAs bearing central mismatches.
View Article and Find Full Text PDFInsufficient tracking of virus introduction, spread, and new lineage emergence for the human monkeypox (mpox) virus 1 (hMPXV1) outbreak of 2022 hindered epidemiological studies and public health response. hMPXV1 mutations accumulated unexpectedly faster than predicted. Thus, new variants with altered pathogenicity could emerge and spread without early detection.
View Article and Find Full Text PDFRibonucleoproteins (RNPs) comprise one or more RNA and protein molecules that interact to form a stable complex, which commonly involves conformational changes in the more flexible RNA components. Here, we propose that Cas12a RNP assembly with its cognate CRISPR RNA (crRNA) guide instead proceeds primarily through Cas12a conformational changes during binding to more stable, prefolded crRNA 5' pseudoknot handles. Phylogenetic reconstructions and sequence and structure alignments revealed that the Cas12a proteins are divergent in sequence and structure while the crRNA 5' repeat region, which folds into a pseudoknot and anchors binding to Cas12a, is highly conserved.
View Article and Find Full Text PDFShort hairpin RNAs, or short hairpin RNAs (shRNAs), are a proven tool for gene knockdown and a promising therapeutic approach for suppression of disease-associated genes. The efficient preparation of shRNA-expressing vectors can sometimes become a bottleneck due to the complexity of shRNA hairpin sequence and structure, especially for repetitive or high GC-content targets. Here, we present improved shRNA cloning and validation methods that enabled efficient and rapid cloning of several shRNAs targeting disease-associated repeat expansions, including GGGGCC, CAG, CTG, CCTG, and CGG into modified pLKO.
View Article and Find Full Text PDFIn late 2019, a novel coronavirus began spreading in Wuhan, China, causing a potentially lethal respiratory viral infection. By early 2020, the novel coronavirus, called SARS-CoV-2, had spread globally, causing the COVID-19 pandemic. The infection and mutation rates of SARS-CoV-2 make it amenable to tracking introduction, spread and evolution by viral genome sequencing.
View Article and Find Full Text PDFACS Appl Mater Interfaces
November 2020
Flexible and ultrasensitive biosensing platforms capable of detecting a large number of trinucleotide repeats (TNRs) are crucial for future technology development needed to combat a variety of genetic disorders. For example, trinucleotide CGG repeat expansions in the gene can cause Fragile X syndrome (FXS) and Fragile X-associated tremor/ataxia syndrome (FXTAS). Current state-of-the-art technologies to detect repeat sequences are expensive, while relying on complicated procedures, and prone to false negatives.
View Article and Find Full Text PDFApproximately 3% of the human genome is composed of short tandem repeat (STR) DNA sequence known as microsatellites, which can be found in both coding and non-coding regions. When associated with genic regions, expansion of microsatellite repeats beyond a critical threshold causes dozens of neurological repeat expansion disorders. To better understand the molecular pathology of repeat expansion disorders, precise cloning of microsatellite repeat sequence and expansion size is highly valuable.
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