RESUMEN
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.
Asunto(s)
Edición Génica , ARN Guía de Kinetoplastida , Animales , Sistemas CRISPR-Cas/genética , Endonucleasas/genética , Endonucleasas/metabolismo , Edición Génica/métodos , Mamíferos/metabolismo , ARN/genética , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismoRESUMEN
The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of organisms. Here we reveal the underlying mechanisms of a third, fundamentally distinct RNA-guided genome-editing platform named CRISPR-CasX, which uses unique structures for programmable double-stranded DNA binding and cleavage. Biochemical and in vivo data demonstrate that CasX is active for Escherichia coli and human genome modification. Eight cryo-electron microscopy structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and a domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a.
Asunto(s)
Proteínas Asociadas a CRISPR/clasificación , Proteínas Asociadas a CRISPR/ultraestructura , Sistemas CRISPR-Cas/genética , Edición Génica , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , Microscopía por Crioelectrón , ADN/química , ADN/metabolismo , ADN/ultraestructura , División del ADN , Escherichia coli/genética , Evolución Molecular , Silenciador del Gen , Genoma Bacteriano/genética , Genoma Humano/genética , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Dominios Proteicos , ARN Guía de Kinetoplastida/metabolismoRESUMEN
In this Article, owing to issues with the first 30 nucleotides of the sgRNA, which run in the opposite direction, corrections have been made to the Protein Data Bank (PDB) accessions in the 'Data availability' section, and this also affects Figs. 3, 4, Extended Data Fig. 6, Supplementary Table 1 and Supplementary Video 1. The original Article has been corrected online. See the accompanying Amendment for further details.
RESUMEN
The prolyl isomerase Pin1 catalyzes the cis-trans isomerization of proline peptide bonds, a non-covalent post-translational modification that influences cellular and molecular processes, including protein-protein interactions. Pin1 is a two-domain enzyme containing a WW domain that recognizes phosphorylated serine/threonine-proline (pS/pT-P) canonical motifs and an enzymatic PPIase domain that catalyzes proline cis-trans isomerization of pS/pT-P motifs. Here, we show that Pin1 uses a tethering mechanism to bind and catalyze proline cis-trans isomerization of a noncanonical motif in the disordered N-terminal activation function-1 (AF-1) domain of the human nuclear receptor PPARγ. NMR reveals multiple Pin1 binding regions within the PPARγ AF-1, including a canonical motif that when phosphorylated by the kinase ERK2 (pS112-P113) binds the Pin1 WW domain with high affinity. NMR methods reveal that Pin1 also binds and accelerates cis-trans isomerization of a noncanonical motif containing a tryptophan-proline motif (W39-P40) previously shown to be involved in an interdomain interaction with the C-terminal ligand-binding domain (LBD). Cellular transcription studies combined with mutagenesis and Pin1 inhibitor treatment reveal a functional role for Pin1-mediated acceleration of cis-trans isomerization of the W39-P40 motif. Our data inform a refined model of the Pin1 catalytic mechanism where the WW domain binds a canonical pS/T-P motif and tethers Pin1 to the target, which enables the PPIase domain to exert catalytic cis-trans isomerization at a distal noncanonical site.