RESUMEN
The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.
Asunto(s)
Sistemas CRISPR-Cas/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Edición Génica/métodos , Proteínas Asociadas a CRISPR/química , ADN/química , Genoma , Modelos Moleculares , ARN/química , ARN Guía de Kinetoplastida/genéticaRESUMEN
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.