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The DNA-PKcs inhibitor AZD7648 enhances CRISPR-Cas9-directed homology-directed repair efficiencies, with potential for clinical utility, but its possible on-target consequences are unknown. We found that genome editing with AZD7648 causes frequent kilobase-scale and megabase-scale deletions, chromosome arm loss and translocations. These large-scale chromosomal alterations evade detection through typical genome editing assays, prompting caution in deploying AZD7648 and reinforcing the need to investigate multiple types of potential editing outcomes., Competing Interests: Competing interests: J.E.C. is a co-founder and scientific advisory board (SAB) member of Spotlight Therapeutics and Serac Biosciences and an SAB member of Mission Therapeutics, Relation Therapeutics, Hornet Bio, Kano Therapeutics and the Joint AstraZeneca–CRUK Functional Genomics Centre. The laboratory of J.E.C. has funded collaborations with Allogene, Cimeio, CSL Behring and Serac. None of these collaborations is related to this paper. T.C. is an advisor to Cimeio Therapeutics, Excision BioTherapeutics, GenCC and Novo Nordisk. T.C. has sponsored research collaborations with Cellectis. T.C. holds a patent on CAST-seq (US11319580B2). F.A., C.M., M.B. and C.V. are employees of CSL Behring AG. The other authors declare no competing interests., (© 2024. The Author(s).)

The dystrophin-glycoprotein-complex (DGC), anchored by the transmembrane protein dystroglycan, functions to mechanically link the extracellular matrix and actin cytoskeleton. Breaking this connection is associated with diseases such as muscular dystrophy, yet cleavage of dystroglycan by matrix-metalloproteinases (MMPs) remains an understudied mechanism to disrupt the DGC. We determined the crystal structure of the membrane-adjacent domain (amino acids 491-722) of E. coli expressed human dystroglycan to understand MMP cleavage regulation. The structural model includes tandem immunoglobulin-like (IGL) and sperm/enterokinase/agrin-like (SEAL) domains, which support proteolysis in diverse receptors to facilitate mechanotransduction, membrane protection, and viral entry. The structure reveals a C-terminal extension that buries the MMP site by packing into a hydrophobic pocket, a unique mechanism of MMP cleavage regulation. We further demonstrate structure-guided and disease-associated mutations disrupt proteolytic regulation using a cell-surface proteolysis assay. Thus disrupted proteolysis is a potentially relevant mechanism for "breaking" the DGC link to contribute to disease pathogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

CRISPR-Cas induced homology-directed repair (HDR) enables the installation of a broad range of precise genomic modifications from an exogenous donor template. However, applications of HDR in human cells are often hampered by poor efficiency, stemming from a preference for error-prone end joining pathways that yield short insertions and deletions. Here, we describe Recursive Editing, an HDR improvement strategy that selectively retargets undesired indel outcomes to create additional opportunities to produce the desired HDR allele. We introduce a software tool, named REtarget, that enables the rational design of Recursive Editing experiments. Using REtarget-designed guide RNAs in single editing reactions, Recursive Editing can simultaneously boost HDR efficiencies and reduce undesired indels. We also harness REtarget to generate databases for particularly effective Recursive Editing sites across the genome, to endogenously tag proteins, and to target pathogenic mutations. Recursive Editing constitutes an easy-to-use approach without potentially deleterious cell manipulations and little added experimental burden., (© 2022. The Author(s).)

Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Adeno-associated virus (AAV) has emerged as a viral gene delivery vector that is safe in humans, able to infect both dividing and arrested cells and drive long-term expression (>6 months). Unfortunately, the naturally evolved properties of many AAV serotypes-including low cell type specificity and largely overlapping tropism-are mismatched to applications that require cell type-specific infection, such as neural circuit mapping or precision gene therapy. A variety of approaches to redirect AAV tropism exist, but there is still the need for a universal solution for directing AAV tropism toward user-defined cellular receptors that does not require extensive case-by-case optimization and works with readily available components. Here, we report AAV engineering approaches that enable programmable receptor-mediated gene delivery. First, we genetically encode small targeting scaffolds into a variable region of an AAV capsid and show that this redirects tropism toward the receptor recognized by these targeting scaffolds and also renders this AAV variant resistant to neutralizing antibodies present in nonhuman primate serum. We then simplify retargeting of tropism by engineering the same variable loop to encode a HUH tag, which forms a covalent bond to single-stranded DNA oligos conjugated to store-bought antibodies. We demonstrate that retargeting this HUH-AAVs toward different receptors is as simple as "arming" a premade noninfective AAV template with a different antibody in a conjugation process that uses widely available reagents and requires no optimization or extensive purification. Composite antibody-AAV nanoparticles structurally separate tropism and payload encapsulation, allowing each to be engineered independently.

Molecular tension sensors measure piconewton forces experienced by individual proteins in the context of the cellular microenvironment. Current genetically encoded tension sensors use FRET to report on extension of a deformable peptide encoded in a cellular protein of interest. Here, we present the development and characterization of a new type of molecular tension sensor based on bioluminescence resonance energy transfer (BRET), which exhibits more desirable spectral properties and an enhanced dynamic range compared to other molecular tension sensors. Moreover, it avoids many disadvantages of FRET measurements in cells, including autofluorescence, photobleaching, and corrections of direct acceptor excitation. We benchmark the sensor by inserting it into the canonical mechanosensing focal adhesion protein vinculin, observing highly resolved gradients of tensional changes across focal adhesions. We anticipate that the BRET tension sensor will expand the toolkit available to study mechanotransduction at a molecular level and allow potential extension to an in vivo context.

Proteolysis of transmembrane receptors is a critical cellular communication mechanism dysregulated in disease, yet decoding proteolytic regulation mechanisms of hundreds of shed receptors is hindered by difficulties controlling stimuli and unknown fates of cleavage products. Notch proteolytic regulation is a notable exception, where intercellular forces drive exposure of a cryptic protease site within a juxtamembrane proteolytic switch domain to activate transcriptional programs. We created a Synthetic Notch Assay for Proteolytic Switches (SNAPS) that exploits the modularity and unequivocal input/response of Notch proteolysis to screen surface receptors for other putative proteolytic switches. We identify several new proteolytic switches among receptors with structural homology to Notch. We demonstrate SNAPS can detect shedding in chimeras of diverse cell surface receptors, leading to new, testable hypotheses. Finally, we establish the assay can be used to measure modulation of proteolysis by potential therapeutics and offer new mechanistic insights into how DECMA-1 disrupts cell adhesion., Competing Interests: AH, EA, WG No competing interests declared, (© 2019, Hayward et al.)

The CRISPR-Cas9 system is a powerful genome-editing tool in which a guide RNA targets Cas9 to a site in the genome, where the Cas9 nuclease then induces a double-stranded break (DSB). The potential of CRISPR-Cas9 to deliver precise genome editing is hindered by the low efficiency of homology-directed repair (HDR), which is required to incorporate a donor DNA template encoding desired genome edits near the DSB. We present a strategy to enhance HDR efficiency by covalently tethering a single-stranded oligodeoxynucleotide (ssODN) to the Cas9-guide RNA ribonucleoprotein (RNP) complex via a fused HUH endonuclease, thus spatially and temporally co-localizing the DSB machinery and donor DNA. We demonstrate up to a 30-fold enhancement of HDR using several editing assays, including repair of a frameshift and in-frame insertions of protein tags. The improved HDR efficiency is observed in multiple cell types and target loci and is more pronounced at low RNP concentrations., Competing Interests: R.S.H. is a co-founder, shareholder, and consultant of ApoGen Biotechnologies Inc. W.R.G., E.J.A. and K.N.L. have filed a provisional patent on this technology. The remaining authors declare no competing interests.