RESUMO
A compact protein with a size of <1,000 amino acids, the CRISPR-associated protein CasX is a fundamentally distinct RNA-guided nuclease when compared to Cas9 and Cas12a. Although it can induce RNA-guided genome editing in mammalian cells, the activity of CasX is less robust than that of the widely used S. pyogenes Cas9. Here, we show that structural features of two CasX homologs and their guide RNAs affect the R-loop complex assembly and DNA cleavage activity. Cryo-EM-based structural engineering of either the CasX protein or the guide RNA produced two new CasX genome editors (DpbCasX-R3-v2 and PlmCasX-R1-v2) with significantly improved DNA manipulation efficacy. These results advance both the mechanistic understanding of CasX and its application as a genome-editing tool.
Assuntos
Edição de Genes , RNA Guia de Cinetoplastídeos , Animais , Sistemas CRISPR-Cas/genética , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes/métodos , Mamíferos/metabolismo , RNA/genética , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
CasX (also known as Cas12e), a Class 2 CRISPR-Cas system, shows promise in genome editing due to its smaller size compared to the widely used Cas9 and Cas12a. Although the structures of CasX-sgRNA-DNA ternary complexes have been resolved and uncover a distinctive NTSB domain, the dynamic behaviors of CasX are not well characterized. In this study, we employed single-molecule and biochemical assays to investigate the conformational dynamics of two CasX homologs, DpbCasX and PlmCasX, from DNA binding to target cleavage and fragment release. Our results indicate that CasX cleaves the non-target strand and the target strand sequentially with relative irreversible dynamics. The two CasX homologs exhibited different cleavage patterns and specificities. The dynamic characterization of CasX also reveals a PAM-proximal seed region, providing guidance for CasX-based effector design. Further studies elucidate the mechanistic basis for why modification of sgRNA and the NTSB domain can affect its activity. Interestingly, CasX has less effective target search efficiency than Cas9 and Cas12a, potentially accounting for its lower genome editing efficiency. This observation opens a new avenue for future protein engineering.
Assuntos
Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Clivagem do DNA , DNA , Transferência Ressonante de Energia de Fluorescência , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/química , DNA/química , DNA/metabolismo , DNA/genética , Imagem Individual de Molécula/métodos , RNA Guia de Sistemas CRISPR-Cas/química , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Edição de Genes/métodos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Conformação ProteicaRESUMO
Ribozymes are catalytic RNAs with diverse functions including self-splicing and polymerization. This work aims to discover natural ribozymes that behave as hydrolytic and sequence-specific DNA endonucleases, which could be repurposed as DNA manipulation tools. Focused on bacterial group II-C introns, we found that many systems without intron-encoded protein propagate multiple copies in their resident genomes. These introns, named HYdrolytic Endonucleolytic Ribozymes (HYERs), cleaved RNA, single-stranded DNA, bubbled double-stranded DNA (dsDNA), and plasmids in vitro. HYER1 generated dsDNA breaks in the mammalian genome. Cryo-electron microscopy analysis revealed a homodimer structure for HYER1, where each monomer contains a Mg2+-dependent hydrolysis pocket and captures DNA complementary to the target recognition site (TRS). Rational designs including TRS extension, recruiting sequence insertion, and heterodimerization yielded engineered HYERs showing improved specificity and flexibility for DNA manipulation.
Assuntos
Clivagem do DNA , Endonucleases , RNA Catalítico , Animais , Microscopia Crioeletrônica , Endonucleases/química , Endonucleases/genética , Hidrólise , Íntrons , Conformação de Ácido Nucleico , Splicing de RNA , RNA Catalítico/química , RNA Catalítico/genéticaRESUMO
CRISPR-Cas modules serve as the adaptive nucleic acid immune systems for prokaryotes, and provide versatile tools for nucleic acid manipulation in various organisms. Here, we discovered a new miniature type V system, CRISPR-Casπ (Cas12l) (~860 aa), from the environmental metagenome. Complexed with a large guide RNA (~170 nt) comprising the tracrRNA and crRNA, Casπ (Cas12l) recognizes a unique 5' C-rich PAM for DNA cleavage under a broad range of biochemical conditions, and generates gene editing in mammalian cells. Cryo-EM study reveals a 'bracelet' architecture of Casπ effector encircling the DNA target at 3.4 Å resolution, substantially different from the canonical 'two-lobe' architectures of Cas12 and Cas9 nucleases. The large guide RNA serves as a 'two-arm' scaffold for effector assembly. Our study expands the knowledge of DNA targeting mechanisms by CRISPR effectors, and offers an efficient but compact platform for DNA manipulation.
Assuntos
DNA , Edição de Genes , DNA/genética , Endonucleases/genética , Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-CasRESUMO
Solute carrier (SLC) transporters meditate many essential physiological functions, including nutrient uptake, ion influx/efflux, and waste disposal. In its protective role against tumors and infections, the mammalian immune system coordinates complex signals to support the proliferation, differentiation, and effector function of individual cell subsets. Recent research in this area has yielded surprising findings on the roles of solute carrier transporters, which were discovered to regulate lymphocyte signaling and control their differentiation, function, and fate by modulating diverse metabolic pathways and balanced levels of different metabolites. In this review, we present current information mainly on glucose transporters, amino-acid transporters, and metal ion transporters, which are critically important for mediating immune cell homeostasis in many different pathological conditions.