CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification.

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.

CasX enzymes comprise a distinct family of RNA-guided genome editors.

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.

Author Correction: CasX enzymes comprise a distinct family of RNA-guided genome editors.

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.

Medial orbitofrontal neurotrophin systems integrate hippocampal input into outcome-specific value representations.

In everyday life, we mentally represent possible consequences of our behaviors and integrate specific outcome values into existing knowledge to inform decisions. The medial orbitofrontal cortex (MO) is necessary to adapt behaviors when outcomes are not immediately available-when they and their values need to be envisioned. Nevertheless, neurobiological mechanisms remain unclear. We find that the neuroplasticity-associated neurotrophin receptor tropomyosin receptor kinase B (TrkB) is necessary for mice to integrate outcome-specific value information into choice behavior. This function appears attributable to memory updating (and not retrieval) and the stabilization of dendritic spines on excitatory MO neurons, which led us to investigate inputs to the MO. Ventral hippocampal (vHC)-to-MO projections appear conditionally necessary for value updating, involved in long-term aversion-based value memory updating. Furthermore, vHC-MO-mediated control of choice is TrkB dependent. Altogether, we reveal a vHC-MO connection by which specific value memories are updated, and we position TrkB within this functional circuit.