CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.

RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ~20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA engineering begins with local helix distortion by transient CRISPR-Cas protein binding.

Mechanism-guided engineering of a minimal biological particle for genome editing.

The widespread application of genome editing to treat or even cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped Delivery Vehicles (EDVs) are engineered virally-derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication components in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs, and demonstrated increased editing in cell lines and therapeutically-relevant primary human T cells. These results show that virally-derived particles can be streamlined to create efficacious genome editing delivery vehicles that could simplify production and manufacturing.

A roadmap for affordable genetic medicines.

Nineteen genetic therapies have been approved by the U.S. Food and Drug Administration (FDA) to date, a number that now includes the first CRISPR genome editing therapy for sickle cell disease, CASGEVY (exagamglogene autotemcel). This extraordinary milestone is widely celebrated because of the promise for future genome editing treatments of previously intractable genetic disorders and cancers. At the same time, such genetic therapies are the most expensive drugs on the market, with list prices exceeding $4 million per patient. Although all approved cell and gene therapies trace their origins to academic or government research institutions, reliance on for-profit pharmaceutical companies for subsequent development and commercialization results in prices that prioritize recouping investments, paying for candidate product failures, and meeting investor and shareholder expectations. To increase affordability and access, sustainable discovery-to-market alternatives are needed that address system-wide deficiencies. Here, we present recommendations of a multi-disciplinary task force assembled to chart such a path. We describe a pricing structure that, once implemented, could reduce per-patient cost tenfold and propose a business model that distributes responsibilities while leveraging diverse funding sources. We also outline how academic licensing provisions, manufacturing innovation and supportive regulations can reduce cost and enable broader patient treatment.

Structure-guided discovery of ancestral CRISPR-Cas13 ribonucleases.

The RNA-guided ribonuclease CRISPR-Cas13 enables adaptive immunity in bacteria and programmable RNA manipulation in heterologous systems. Cas13s share limited sequence similarity, hindering discovery of related or ancestral systems. To address this, we developed an automated structural-search pipeline to identify an ancestral clade of Cas13 (Cas13an) and further trace Cas13 origins to defense-associated ribonucleases. Despite being one-third the size of other Cas13s, Cas13an mediates robust programmable RNA depletion and defense against diverse bacteriophages. However, unlike its larger counterparts, Cas13an uses a single active site for both CRISPR RNA processing and RNA-guided cleavage, revealing that the ancestral nuclease domain has two modes of activity. Discovery of Cas13an deepens our understanding of CRISPR-Cas evolution and expands opportunities for precision RNA editing, showcasing the promise of structure-guided genome mining.

Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes.

Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage, compared to just days in isogenic iPSCs. Furthermore, Cas9-treated neurons upregulated unexpected DNA repair genes, including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing.

Single-molecule live-cell RNA imaging with CRISPR-Csm.

High-resolution, real-time imaging of RNA is essential for understanding the diverse, dynamic behaviors of individual RNA molecules in single cells. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR-Csm complex with multiplexed guide RNAs for efficient, direct visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we tracked individual endogenous NOTCH2 and MAP1B mRNA transcripts in living cells and identified two distinct localization mechanisms: co-translational translocation of NOTCH2 mRNA at the endoplasmic reticulum, and directional transport of MAP1B mRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.

Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9.

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

RNA language models predict mutations that improve RNA function.

Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. While advances in deep learning enable the prediction of accurate protein structural models, RNA structure prediction is not possible at present due to a lack of abundant high-quality reference data. Furthermore, available sequence data are generally not associated with organismal phenotypes that could inform RNA function. We created GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB). GARNET links RNA sequences derived from GTDB genomes to experimental and predicted optimal growth temperatures of GTDB reference organisms. This enables construction of deep and diverse RNA sequence alignments to be used for machine learning. Using GARNET, we define the minimal requirements for a sequence- and structure-aware RNA generative model. We also develop a GPT-like language model for RNA in which triplet tokenization provides optimal encoding. Leveraging hyperthermophilic RNAs in GARNET and these RNA generative models, we identified mutations in ribosomal RNA that confer increased thermostability to the Escherichia coli ribosome. The GTDB-derived data and deep learning models presented here provide a foundation for understanding the connections between RNA sequence, structure, and function.

An essential and highly selective protein import pathway encoded by nucleus-forming phage.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here we identify two components of this novel protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA, that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together our results allow us to propose a multistep model for the Protein Import Chimallivirus (PIC) pathway, where proteins are targeted to PicA by amino acids on their surface, and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely-related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts. Significance Statement: The phage nucleus is an enclosed replication compartment built by Chimalliviridae phages that, similar to the eukaryotic nucleus, separates transcription from translation and selectively imports certain proteins. This allows the phage to concentrate proteins required for DNA replication and transcription while excluding DNA-targeting host defense proteins. However, the mechanism of selective trafficking into the phage nucleus is currently unknown. Here we determine the region of a phage nuclear protein that targets it for nuclear import and identify a conserved, essential nuclear shell-associated protein that plays a key role in this process. This work provides the first mechanistic model of selective import into the phage nucleus.

An essential and highly selective protein import pathway encoded by nucleus-forming phage.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

Hachiman is a genome integrity sensor.

Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman comprises a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease. HamB is the sensor helicase. HamB constrains HamA activity during surveillance of intact dsDNA. When the HamAB complex detects DNA damage, HamB helicase activity liberates HamA, unleashing nuclease activity. Hachiman activation degrades all DNA in the cell, creating 'phantom' cells devoid of both phage and host DNA. We demonstrate Hachiman activation in the absence of phage by treatment with DNA-damaging agents, suggesting that Hachiman responds to aberrant DNA states. Phylogenetic similarities between the Hachiman helicase and eukaryotic enzymes suggest this bacterial immune system has been repurposed for diverse functions across all domains of life.

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

Targeted nonviral delivery of genome editors in vivo.

Cell-type-specific in vivo delivery of genome editing molecules is the next breakthrough that will drive biological discovery and transform the field of cell and gene therapy. Here, we discuss recent advances in the delivery of CRISPR-Cas genome editors either as preassembled ribonucleoproteins or encoded in mRNA. Both strategies avoid pitfalls of viral vector-mediated delivery and offer advantages including transient editor lifetime and potentially streamlined manufacturing capability that are already proving valuable for clinical use. We review current applications and future opportunities of these emerging delivery approaches that could make genome editing more efficacious and accessible in the future.

Engineering self-deliverable ribonucleoproteins for genome editing in the brain.

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects. However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineer self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identifies potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins establishes a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibits substantially improved editing efficacy compared to other constructs. We find that self-deliverable Cas9 RNPs generate robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editing in vitro and in vivo.

In vivo human T cell engineering with enveloped delivery vehicles.

Viruses and virally derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Here, we show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR-Cas9 protein and guide RNA can deliver genome editing tools to specific cells. Compared to conventional vectors like adeno-associated virus that rely on evolved capsid tropisms to deliver virally encoded cargo, these Cas9-packaging enveloped delivery vehicles (Cas9-EDVs) leverage predictable antibody-antigen interactions to transiently deliver genome editing machinery selectively to cells of interest. Antibody-targeted Cas9-EDVs preferentially confer genome editing in cognate target cells over bystander cells in mixed populations, both ex vivo and in vivo. By using multiplexed targeting molecules to direct delivery to human T cells, Cas9-EDVs enable the generation of genome-edited chimeric antigen receptor T cells in humanized mice, establishing a programmable delivery modality with the potential for widespread therapeutic utility.

Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-) protein into ribonucleoprotein particles (RNPs), 38±10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining mutant proteins in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multi-valent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

Targeting the non-coding genome and temozolomide signature enables CRISPR-mediated glioma oncolysis.

Glioblastoma (GBM) is the most common lethal primary brain cancer in adults. Despite treatment regimens including surgical resection, radiotherapy, and temozolomide (TMZ) chemotherapy, growth of residual tumor leads to therapy resistance and death. At recurrence, a quarter to a third of all gliomas have hypermutated genomes, with mutational burdens orders of magnitude greater than in normal tissue. Here, we quantified the mutational landscape progression in a patient's primary and recurrent GBM, and we uncovered Cas9-targetable repeat elements. We show that CRISPR-mediated targeting of highly repetitive loci enables rapid elimination of GBM cells, an approach we term "genome shredding." Importantly, in the patient's recurrent GBM, we identified unique repeat sequences with TMZ mutational signature and demonstrated that their CRISPR targeting enables cancer-specific cell ablation. "Cancer shredding" leverages the non-coding genome and therapy-induced mutational signatures for targeted GBM cell depletion and provides an innovative paradigm to develop treatments for hypermutated glioma.

Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes.

RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and a TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12s. In eukaryotes, TnpB homologs occur as two distinct types, Fanzor1s and Fanzor2s. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, which revealed that both Fanzor1s and Fanzor2s stem from a single lineage of IS607 TnpBs with unusual active site arrangement. The widespread nature of Fanzors implies that the properties of this particular lineage of IS607 TnpBs were particularly suited to adaptation in eukaryotes. Biochemical analysis of an IS607 TnpB and Fanzor1s revealed common strategies employed by TnpBs and Fanzors to co-evolve with their cognate transposases. Collectively, our results provide a new model of sequential evolution from IS607 TnpBs to Fanzor2s, and Fanzor2s to Fanzor1s that details how genes of prokaryotic origin evolve to give rise to new protein families in eukaryotes.