Exploring the potential of cell-derived vesicles for transient delivery of gene editing payloads.

Gene editing technologies have the potential to correct genetic disorders by modifying, inserting, or deleting specific DNA sequences or genes, paving the way for a new class of genetic therapies. While gene editing tools continue to be improved to increase their precision and efficiency, the limited efficacy of in vivo delivery remains a major hurdle for clinical use. An ideal delivery vehicle should be able to target a sufficient number of diseased cells in a transient time window to maximize on-target editing and mitigate off-target events and immunogenicity. Here, we review major advances in novel delivery platforms based on cell-derived vesicles - extracellular vesicles and virus-like particles - for transient delivery of gene editing payloads. We discuss major findings regarding packaging, in vivo biodistribution, therapeutic efficacy, and safety concerns of cell-derived vesicles delivery of gene-editing cargos and their potential for clinical translation.

CRISPR-Cas Genome Editing in Ex Vivo Human Lungs to Rewire the Translational Path of Genome-Targeting Therapeutics.

The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both in vivo and ex vivo organ genome-editing therapeutics. One of the promising applications is to genetically modify donor organs prior to implantation. The implantation of optimized donor organs with long-lasting immunomodulatory capacity holds promise for reducing the need for lifelong potent whole-body immunosuppression in recipients. However, assessing genome-targeting interventions in a clinically relevant manner prior to clinical trials remains a major challenge owing to the limited modalities available. This study introduces a novel platform for testing genome editing in human lungs ex vivo, effectively simulating preimplantation genetic engineering of donor organs. We identified gene regulatory elements whose disruption via Cas nucleases led to the upregulation of the immunomodulatory gene interleukin 10 (IL-10). We combined this approach with adenoviral vector-mediated IL-10 delivery to create favorable kinetics for early (immediate postimplantation) graft immunomodulation. Using ex vivo organ machine perfusion and precision-cut tissue slice technology, we demonstrated the feasibility of evaluating CRISPR genome editing in human lungs. To overcome the assessment limitations in ex vivo perfused human organs, we conducted an in vivo rodent study and demonstrated both early gene induction and sustained editing of the lung. Collectively, our findings lay the groundwork for a first-in-human-organ study to overcome the current translational barriers of genome-targeting therapeutics.

Splice modulators target PMS1 to reduce somatic expansion of the Huntington's disease-associated CAG repeat.

Huntington's disease (HD) is a dominant neurological disorder caused by an expanded HTT exon 1 CAG repeat that lengthens huntingtin's polyglutamine tract. Lowering mutant huntingtin has been proposed for treating HD, but genetic modifiers implicate somatic CAG repeat expansion as the driver of onset. We find that branaplam and risdiplam, small molecule splice modulators that lower huntingtin by promoting HTT pseudoexon inclusion, also decrease expansion of an unstable HTT exon 1 CAG repeat in an engineered cell model. Targeted CRISPR-Cas9 editing shows this effect is not due to huntingtin lowering, pointing instead to pseudoexon inclusion in PMS1. Homozygous but not heterozygous inactivation of PMS1 also reduces CAG repeat expansion, supporting PMS1 as a genetic modifier of HD and a potential target for therapeutic intervention. Although splice modulation provides one strategy, genome-wide transcriptomics also emphasize consideration of cell-type specific effects and polymorphic variation at both target and off-target sites.

In situ architecture of Opa1-dependent mitochondrial cristae remodeling.

Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to lack of native three-dimensional views of cristae. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe a variety of cristae shapes with distinct trends dependent on s-Opa1:l-Opa1 balance. Increased l-Opa1 levels promote cristae stacking and elongated mitochondria, while increased s-Opa1 levels correlated with irregular cristae packing and round mitochondria shape. Functional assays indicate a role for l-Opa1 in wild-type apoptotic and calcium handling responses, and show a compromised respiratory function under Opa1 imbalance. In summary, we provide three-dimensional visualization of cristae architecture to reveal relationships between mitochondrial ultrastructure and cellular function dependent on Opa1-mediated membrane remodeling.

Optimization of base editors for the functional correction of SMN2 as a treatment for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by mutations in SMN1. SMN2 is a paralogous gene with a C•G-to-T•A transition in exon 7, which causes this exon to be skipped in most SMN2 transcripts, and results in low levels of the protein survival motor neuron (SMN). Here we show, in fibroblasts derived from patients with SMA and in a mouse model of SMA that, irrespective of the mutations in SMN1, adenosine base editors can be optimized to target the SMN2 exon-7 mutation or nearby regulatory elements to restore the normal expression of SMN. After optimizing and testing more than 100 guide RNAs and base editors, and leveraging Cas9 variants with high editing fidelity that are tolerant of different protospacer-adjacent motifs, we achieved the reversion of the exon-7 mutation via an A•T-to-G•C edit in up to 99% of fibroblasts, with concomitant increases in the levels of the SMN2 exon-7 transcript and of SMN. Targeting the SMN2 exon-7 mutation via base editing or other CRISPR-based methods may provide long-lasting outcomes to patients with SMA.

PAM-flexible genome editing with an engineered chimeric Cas9.

CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN (R = A or G, Y = C or T) PAM preference, with the N-terminus of Sc + +, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse PAMs and disease-related loci for potential therapeutic applications. In total, the approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.

Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases.

Genome editing technologies that install diverse edits can widely enable genetic studies and new therapeutics. Here we develop click editing, a genome writing platform that couples the advantageous properties of DNA-dependent DNA polymerases with RNA-programmable nickases (e.g. CRISPR-Cas) to permit the installation of a range of edits including substitutions, insertions, and deletions. Click editors (CEs) leverage the "click"-like bioconjugation ability of HUH endonucleases (HUHes) with single stranded DNA substrates to covalently tether "click DNA" (clkDNA) templates encoding user-specifiable edits at targeted genomic loci. Through iterative optimization of the modular components of CEs (DNA polymerase and HUHe orthologs, architectural modifications, etc.) and their clkDNAs (template configurations, repair evading substitutions, etc.), we demonstrate the ability to install precise genome edits with minimal indels and no unwanted byproduct insertions. Since clkDNAs can be ordered as simple DNA oligonucleotides for cents per base, it is possible to screen many different clkDNA parameters rapidly and inexpensively to maximize edit efficiency. Together, click editing is a precise and highly versatile platform for modifying genomes with a simple workflow and broad utility across diverse biological applications.

Treatment of monogenic and digenic dominant genetic hearing loss by CRISPR-Cas9 ribonucleoprotein delivery in vivo.

Mutations in Atp2b2, an outer hair cell gene, cause dominant hearing loss in humans. Using a mouse model Atp2b2Obl/+, with a dominant hearing loss mutation (Oblivion), we show that liposome-mediated in vivo delivery of CRISPR-Cas9 ribonucleoprotein complexes leads to specific editing of the Obl allele. Large deletions encompassing the Obl locus and indels were identified as the result of editing. In vivo genome editing promotes outer hair cell survival and restores their function, leading to hearing recovery. We further show that in a double-dominant mutant mouse model, in which the Tmc1 Beethoven mutation and the Atp2b2 Oblivion mutation cause digenic genetic hearing loss, Cas9/sgRNA delivery targeting both mutations leads to partial hearing recovery. These findings suggest that liposome-RNP delivery can be used as a strategy to recover hearing with dominant mutations in OHC genes and with digenic mutations in the auditory hair cells, potentially expanding therapeutics of gene editing to treat hearing loss.

PMS1 as a target for splice modulation to prevent somatic CAG repeat expansion in Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder whose motor, cognitive, and behavioral manifestations are caused by an expanded, somatically unstable CAG repeat in the first exon of HTT that lengthens a polyglutamine tract in huntingtin. Genome-wide association studies (GWAS) have revealed DNA repair genes that influence the age-at-onset of HD and implicate somatic CAG repeat expansion as the primary driver of disease timing. To prevent the consequent neuronal damage, small molecule splice modulators (e.g., branaplam) that target HTT to reduce the levels of huntingtin are being investigated as potential HD therapeutics. We found that the effectiveness of the splice modulators can be influenced by genetic variants, both at HTT and other genes where they promote pseudoexon inclusion. Surprisingly, in a novel hTERT-immortalized retinal pigment epithelial cell (RPE1) model for assessing CAG repeat instability, these drugs also reduced the rate of HTT CAG expansion. We determined that the splice modulators also affect the expression of the mismatch repair gene PMS1, a known modifier of HD age-at-onset. Genome editing at specific HTT and PMS1 sequences using CRISPR-Cas9 nuclease confirmed that branaplam suppresses CAG expansion by promoting the inclusion of a pseudoexon in PMS1, making splice modulation of PMS1 a potential strategy for delaying HD onset. Comparison with another splice modulator, risdiplam, suggests that other genes affected by these splice modulators also influence CAG instability and might provide additional therapeutic targets.

Rescue of hearing by adenine base editing in a humanized mouse model of Usher syndrome type 1F.

Usher syndrome type 1F (USH1F), characterized by congenital lack of hearing and balance and progressive loss of vision, is caused by mutations in the PCDH15 gene. In the Ashkenazi population, a recessive truncation mutation accounts for a large proportion of USH1F cases. The truncation is caused by a single C→T mutation, which converts an arginine codon to a stop (R245X). To test the potential for base editors to revert this mutation, we developed a humanized Pcdh15R245X mouse model for USH1F. Mice homozygous for the R245X mutation were deaf and exhibited profound balance deficits, while heterozygous mice were unaffected. Here we show that an adenine base editor (ABE) is capable of reversing the R245X mutation to restore the PCDH15 sequence and function. We packaged a split-intein ABE into dual adeno-associated virus (AAV) vectors and delivered them into cochleas of neonatal USH1F mice. Hearing was not restored in a Pcdh15 constitutive null mouse despite base editing, perhaps because of early disorganization of cochlear hair cells. However, injection of vectors encoding the split ABE into a late-deletion conditional Pcdh15 knockout rescued hearing. This study demonstrates the ability of an ABE to correct the PCDH15 R245X mutation in the cochlea and restore hearing.

Immunomodulation of the donor lung with CRISPR-mediated activation of IL-10 expression.

BACKGROUND: Inflammatory injury in the donor lung remains a persistent challenge in lung transplantation that limits donor organ usage and post-transplant outcomes. Inducing immunomodulatory capacity in donor organs could address this unsolved clinical problem. We sought to apply clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) technologies to the donor lung to fine-tune immunomodulatory gene expression, exploring for the first time the therapeutic use of CRISPR-mediated transcriptional activation in the whole donor lung. METHODS: We explored the feasibility of CRISPR-mediated transcriptional upregulation of interleukin 10 (IL-10), a key immunomodulatory cytokine, in vitro and in vivo. We first evaluated the potency, titratability, and multiplexibility of the gene activation in rat and human cell lines. Next, in vivo CRISPR-mediated IL-10 activation was characterized in rat lungs. Finally, the IL-10-activated donor lungs were transplanted into recipient rats to assess the feasibility in a transplant setting. RESULTS: The targeted transcriptional activation induced robust and titrable IL-10 upregulation in vitro. The combination of guide RNAs also facilitated multiplex gene modulation, that is, simultaneous activation of IL-10 and IL1 receptor antagonist. In vivo profiling demonstrated that adenoviral delivery of Cas9-based activators to the lung was feasible with the use of immunosuppression, which is routinely applied to organ transplant recipients. The transcriptionally modulated donor lungs retained IL-10 upregulation in isogeneic and allogeneic recipients. CONCLUSIONS: Our findings highlight the potential of CRISPR epigenome editing to improve lung transplant outcomes by creating a more favorable immunomodulatory environment in the donor organ, a paradigm that may be extendable to other organ transplants.

Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington's disease.

An expanded CAG repeat in the huntingtin gene ( HTT ) causes Huntington's disease (HD). Since the length of uninterrupted CAG repeat, not polyglutamine, determines the age-at-onset in HD, base editing strategies to convert CAG to CAA are anticipated to delay onset by shortening the uninterrupted CAG repeat. Here, we developed base editing strategies to convert CAG in the repeat to CAA and determined their molecular outcomes and effects on relevant disease phenotypes. Base editing strategies employing combinations of cytosine base editors and gRNAs efficiently converted CAG to CAA at various sites in the CAG repeat without generating significant indels, off-target edits, or transcriptome alterations, demonstrating their feasibility and specificity. Candidate BE strategies converted CAG to CAA on both expanded and non-expanded CAG repeats without altering HTT mRNA and protein levels. In addition, somatic CAG repeat expansion, which is the major disease driver in HD, was significantly decreased by a candidate BE strategy treatment in HD knock-in mice carrying canonical CAG repeats. Notably, CAG repeat expansion was abolished entirely in HD knock-in mice carrying CAA-interrupted repeats, supporting the therapeutic potential of CAG-to-CAA conversion base editing strategies in HD and potentially other repeat expansion disorders.

Interleukin-3 coordinates glial-peripheral immune crosstalk to incite multiple sclerosis.

Glial cells and central nervous system (CNS)-infiltrating leukocytes contribute to multiple sclerosis (MS). However, the networks that govern crosstalk among these ontologically distinct populations remain unclear. Here, we show that, in mice and humans, CNS-resident astrocytes and infiltrating CD44hiCD4+ T cells generated interleukin-3 (IL-3), while microglia and recruited myeloid cells expressed interleukin-3 receptor-ɑ (IL-3Rɑ). Astrocytic and T cell IL-3 elicited an immune migratory and chemotactic program by IL-3Rɑ+ myeloid cells that enhanced CNS immune cell infiltration, exacerbating MS and its preclinical model. Multiregional snRNA-seq of human CNS tissue revealed the appearance of IL3RA-expressing myeloid cells with chemotactic programming in MS plaques. IL3RA expression by plaque myeloid cells and IL-3 amount in the cerebrospinal fluid predicted myeloid and T cell abundance in the CNS and correlated with MS severity. Our findings establish IL-3:IL-3RA as a glial-peripheral immune network that prompts immune cell recruitment to the CNS and worsens MS.

PAM-Flexible Genome Editing with an Engineered Chimeric Cas9.

CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN PAM preference, with the N-terminus of Sc++, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse NNN PAMs and disease-related loci for potential therapeutic applications. In total, the unique approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.

Multimodal CRISPR perturbations of GWAS loci associated with coronary artery disease in vascular endothelial cells.

Genome-wide association studies have identified >250 genetic variants associated with coronary artery disease (CAD), but the causal variants, genes and molecular mechanisms remain unknown at most loci. We performed pooled CRISPR screens to test the impact of sequences at or near CAD-associated genetic variants on vascular endothelial cell functions. Using CRISPR knockout, inhibition and activation, we targeted 1998 variants at 83 CAD loci to assess their effect on three adhesion proteins (E-selectin, ICAM1, VCAM1) and three key endothelial functions (nitric oxide and reactive oxygen species production, calcium signalling). At a false discovery rate ≤10%, we identified significant CRISPR perturbations near 42 variants located within 26 CAD loci. We used base editing to validate a putative causal variant in the promoter of the FES gene. Although a few of the loci include genes previously characterized in endothelial cells (e.g. AIDA, ARHGEF26, ADAMTS7), most are implicated in endothelial dysfunction for the first time. Detailed characterization of one of these new loci implicated the RNA helicase DHX38 in vascular endothelial cell senescence. While promising, our results also highlighted several limitations in using CRISPR perturbations to functionally dissect GWAS loci, including an unknown false negative rate and potential off-target effects.

Sniper2L is a high-fidelity Cas9 variant with high activity.

Although several high-fidelity SpCas9 variants have been reported, it has been observed that this increased specificity is associated with reduced on-target activity, limiting the applications of the high-fidelity variants when efficient genome editing is required. Here, we developed an improved version of Sniper-Cas9, Sniper2L, which represents an exception to this trade-off trend as it showed higher specificity with retained high activity. We evaluated Sniper2L activities at a large number of target sequences and developed DeepSniper, a deep learning model that can predict the activity of Sniper2L. We also confirmed that Sniper2L can induce highly efficient and specific editing at a large number of target sequences when it is delivered as a ribonucleoprotein complex. Mechanically, the high specificity of Sniper2L originates from its superior ability to avoid unwinding a target DNA containing even a single mismatch. We envision that Sniper2L will be useful when efficient and specific genome editing is required.

In situ architecture of Opa1-dependent mitochondrial cristae remodeling.

Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to lack of native three-dimensional views of cristae. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe a variety of cristae shapes with distinct trends dependent on s-Opa1:l-Opa1 balance. Increased l-Opa1 levels promote cristae stacking and elongated mitochondria while increased s-Opa1 levels correlated with irregular cristae packing and round mitochondria shape. Functional assays indicate a role for l-Opa1 in wild-type apoptotic and calcium handling responses, and compromised respiratory function under Opa1 imbalance. In summary, we provide three-dimensional visualization of cristae architecture to reveal relationships between mitochondrial ultrastructure and cellular function dependent on Opa1-mediated membrane remodeling.

Base editing as a genetic treatment for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the SMN1 gene. Despite the development of various therapies, outcomes can remain suboptimal in SMA infants and the duration of such therapies are uncertain. SMN2 is a paralogous gene that mainly differs from SMN1 by a C•G-to-T•A transition in exon 7, resulting in the skipping of exon 7 in most SMN2 transcripts and production of only low levels of survival motor neuron (SMN) protein. Genome editing technologies targeted to the SMN2 exon 7 mutation could offer a therapeutic strategy to restore SMN protein expression to normal levels irrespective of the patient SMN1 mutation. Here, we optimized a base editing approach to precisely edit SMN2, reverting the exon 7 mutation via an A•T-to-G•C base edit. We tested a range of different adenosine base editors (ABEs) and Cas9 enzymes, resulting in up to 99% intended editing in SMA patient-derived fibroblasts with concomitant increases in SMN2 exon 7 transcript expression and SMN protein levels. We generated and characterized ABEs fused to high-fidelity Cas9 variants which reduced potential off-target editing. Delivery of these optimized ABEs via dual adeno-associated virus (AAV) vectors resulted in precise SMN2 editing in vivo in an SMA mouse model. This base editing approach to correct SMN2 should provide a long-lasting genetic treatment for SMA with advantages compared to current nucleic acid, small molecule, or exogenous gene replacement therapies. More broadly, our work highlights the potential of PAMless SpRY base editors to install edits efficiently and safely.

Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR-Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.

Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases.

CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. However, the utility of type V-K CASTs is hindered by high off-target integration and a transposition mechanism that results in a mixture of desired simple cargo insertions and undesired plasmid cointegrate products. Here we overcome both limitations by engineering new CASTs with improved integration product purity and genome-wide specificity. To do so, we engineered a nicking homing endonuclease fusion to TnsB (named HELIX) to restore the 5' nicking capability needed for cargo excision on the DNA donor. HELIX enables cut-and-paste DNA insertion with up to 99.4% simple insertion product purity, while retaining robust integration efficiencies on genomic targets. HELIX has substantially higher on-target specificity than canonical CASTs, and we identify several novel factors that further regulate targeted and genome-wide integration. Finally, we extend HELIX to other type V-K orthologs and demonstrate the feasibility of HELIX-mediated integration in human cell contexts.