RESUMEN
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 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 NNN PAMs and disease-related loci for potential therapeutic applications. In total, the unique approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.
RESUMEN
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. In total, the approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , GenomaRESUMEN
The novel coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) continues to pose a substantial global health threat. Along with vaccines and targeted therapeutics, there is a critical need for rapid diagnostic solutions. In this work, we use computational protein modeling tools to suggest molecular beacon architectures that function as conformational switches for high-sensitivity detection of the SARS-CoV-2 spike protein receptor binding domain (S-RBD). Integrating these beacons on a miniaturized total internal reflection fluorescence (mini-TIRF) microscope, we detect the S-RBD and pseudotyped SARS-CoV-2 with limits of detection in the femtomolar range. We envision that our designed mini-TIRF platform will serve as a robust platform for point-of-care diagnostics for SARS-CoV-2 and future emergent viral threats.
RESUMEN
The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has elicited a global health crisis of catastrophic proportions. With only a few vaccines approved for early or limited use, there is a critical need for effective antiviral strategies. In this study, we report a unique antiviral platform, through computational design of ACE2-derived peptides which both target the viral spike protein receptor binding domain (RBD) and recruit E3 ubiquitin ligases for subsequent intracellular degradation of SARS-CoV-2 in the proteasome. Our engineered peptide fusions demonstrate robust RBD degradation capabilities in human cells and are capable of inhibiting infection-competent viral production, thus prompting their further experimental characterization and therapeutic development.