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 , Endodesoxirribonucleases , Escherichia coli , Edição de Genes , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , DNA/metabolismo , DNA/genética , DNA/química , CinéticaRESUMO
Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that block Plasmodium parasite development in the mosquito midgut, thus preventing mosquitoes from becoming infectious. While the Pro-domain and first of fourteen 6-Cysteine domains (Pro-D1) of the Plasmodium gamete surface protein Pfs230 are known targets of transmission-blocking antibodies, no studies to date have discovered other Pfs230 domains that are functional targets. Here, we show that a murine monoclonal antibody (mAb), 18F25.1, targets Pfs230 Domain 7. We generated a subclass-switched complement-fixing variant, mAb 18F25.2a, using a CRISPR/Cas9-based hybridoma engineering method. This subclass-switched mAb 18F25.2a induced lysis of female gametes in vitro. Importantly, mAb 18F25.2a potently reduced P. falciparum infection of Anopheles stephensi mosquitoes in a complement-dependent manner, as assessed by standard membrane feeding assays. Together, our data identify Pfs230 Domain 7 as target for transmission-blocking antibodies and provide a strong incentive to study domains outside Pfs230Pro-D1 as TBV candidates.
