Cooperativity between Cas9 and hyperactive AID establishes broad and diversifying mutational footprints in base editors.

The partnership of DNA deaminase enzymes with CRISPR-Cas nucleases is now a well-established method to enable targeted genomic base editing. However, an understanding of how Cas9 and DNA deaminases collaborate to shape base editor (BE) outcomes has been lacking. Here, we support a novel mechanistic model of base editing by deriving a range of hyperactive activation-induced deaminase (AID) base editors (hBEs) and exploiting their characteristic diversifying activity. Our model involves multiple layers of previously underappreciated cooperativity in BE steps including: (i) Cas9 binding can potentially expose both DNA strands for 'capture' by the deaminase, a feature that is enhanced by guide RNA mismatches; (ii) after strand capture, the intrinsic activity of the DNA deaminase can tune window size and base editing efficiency; (iii) Cas9 defines the boundaries of editing on each strand, with deamination blocked by Cas9 binding to either the PAM or the protospacer and (iv) non-canonical edits on the guide RNA bound strand can be further elicited by changing which strand is nicked by Cas9. Leveraging insights from our mechanistic model, we create novel hBEs that can remarkably generate simultaneous C > T and G > A transitions over >65 bp with significant potential for targeted gene diversification.

Efficient engineering of human and mouse primary cells using peptide-assisted genome editing.

Simple, efficient and well-tolerated delivery of CRISPR genome editing systems into primary cells remains a major challenge. Here we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR-Cas system for rapid and robust editing of primary cells with minimal toxicity. The PAGE system requires only a 30-min incubation with a cell-penetrating Cas9 or Cas12a and a cell-penetrating endosomal escape peptide to achieve robust single and multiplex genome editing. Unlike electroporation-based methods, PAGE gene editing has low cellular toxicity and shows no significant transcriptional perturbation. We demonstrate rapid and efficient editing of primary cells, including human and mouse T cells, as well as human hematopoietic progenitor cells, with editing efficiencies upwards of 98%. PAGE provides a broadly generalizable platform for next-generation genome engineering in primary cells.

The RNA helicase DDX39A binds a conserved structure in chikungunya virus RNA to control infection.

Alphaviruses are a large group of re-emerging arthropod-borne RNA viruses. The compact viral RNA genomes harbor diverse structures that facilitate replication. These structures can be recognized by antiviral cellular RNA-binding proteins, including DExD-box (DDX) helicases, that bind viral RNAs to control infection. The full spectrum of antiviral DDXs and the structures that are recognized remain unclear. Genetic screening identified DDX39A as antiviral against the alphavirus chikungunya virus (CHIKV) and other medically relevant alphaviruses. Upon infection, the predominantly nuclear DDX39A accumulates in the cytoplasm inhibiting alphavirus replication, independent of the canonical interferon pathway. Biochemically, DDX39A binds to CHIKV genomic RNA, interacting with the 5' conserved sequence element (5'CSE), which is essential for the antiviral activity of DDX39A. Altogether, DDX39A relocalization and binding to a conserved structural element in the alphavirus genomic RNA attenuates infection, revealing a previously unknown layer to the cellular control of infection.