Publications by authors named "Rachel A Silverstein"
Article Synopsis
- Genetic vascular disorders, particularly multisystemic smooth muscle dysfunction syndrome (MSMDS), result from mutations in the alpha actin isotype 2 gene and can lead to severe health issues such as stroke and early childhood death.
- The research focused on correcting the common R179H mutation using a specially engineered CRISPR-Cas9 enzyme designed for high accuracy, decreasing unintended edits during the gene correction process.
- By utilizing a murine model that mimics human MSMDS symptoms, the study demonstrated that delivering the customized editing tool significantly improved survival and health outcomes in affected mice, indicating potential for lasting treatments in humans.
CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN (R = A or G, Y = C or T) PAM preference, with the N-terminus of Sc + +, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse PAMs and disease-related loci for potential therapeutic applications.
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- CRISPR-Cas9, particularly from Streptococcus pyogenes, is crucial for bacterial immunity but its evolutionary history is not well understood.!
- This study explores the evolution of Cas9 by resurrecting ancient nucleases from extinct firmicutes that existed 2.6 billion years ago and finds they had more flexible RNA and DNA target requirements compared to today's versions.!
- The research reveals that ancient Cas9 enzymes showed a gradual shift in function from nicking to creating double-strand breaks and were effective in editing both DNA and RNA, including in human cells, highlighting an evolutionary path to modern enzymes.
Methods for in vitro DNA cleavage and molecular cloning remain unable to precisely cleave DNA directly adjacent to bases of interest. Restriction enzymes (REs) must bind specific motifs, whereas wild-type CRISPR-Cas9 or CRISPR-Cas12 nucleases require protospacer adjacent motifs (PAMs). Here we explore the utility of our previously reported near-PAMless SpCas9 variant, named SpRY, to serve as a universal DNA cleavage tool for various cloning applications.
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