RESUMO
CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search.
Assuntos
Proteína 9 Associada à CRISPR , Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Edição de Genes , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , DNA/metabolismo , DNA/genética , DNA/química , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Edição de Genes/métodos , Cinética , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismoRESUMO
We investigate and validate a novel method to fabricate ultrathin optical probes for common-path optical coherence tomography (CP-OCT). The probes are obtained using a 65 µm barium titanate microsphere inserted into an inward concave cone chemically etched at the end of a single-mode fiber. We demonstrate that the high refractive index (n=1.95) of the barium titanate microspheres allows one to maintain high sensitivity even while imaging in liquids, reaching a sensitivity of 83 dB. Due to its low cost, flexibility, and ease of use, the probe holds promise for the development of a new generation of ultrathin needle-based OCT systems.