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.

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.

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.

Precise DNA cleavage using CRISPR-SpRYgests.

Methods for in vitro DNA cleavage and molecular cloning remain unable to precisely cleave DNA directly adjacent to bases of interest. Restriction enzymes (REs) must bind specific motifs, whereas wild-type CRISPR-Cas9 or CRISPR-Cas12 nucleases require protospacer adjacent motifs (PAMs). Here we explore the utility of our previously reported near-PAMless SpCas9 variant, named SpRY, to serve as a universal DNA cleavage tool for various cloning applications. By performing SpRY DNA digests (SpRYgests) using more than 130 guide RNAs (gRNAs) sampling a wide diversity of PAMs, we discovered that SpRY is PAMless in vitro and can cleave DNA at practically any sequence, including sites refractory to cleavage with wild-type SpCas9. We illustrate the versatility and effectiveness of SpRYgests to improve the precision of several cloning workflows, including those not possible with REs or canonical CRISPR nucleases. We also optimize a rapid and simple one-pot gRNA synthesis protocol to streamline SpRYgest implementation. Together, SpRYgests can improve various DNA engineering applications that benefit from precise DNA breaks.

Making the cut with PAMless CRISPR-Cas enzymes.

Genome editing technologies simplify our ability to rewrite genetic blueprints of life. However, CRISPR-Cas enzymes found in nature can only manipulate a fraction of the genome. To overcome this limitation, new Cas variants have been developed that unlock nearly the entire genome for editing.

Astrocytic interleukin-3 programs microglia and limits Alzheimer's disease.

Communication within the glial cell ecosystem is essential for neuronal and brain health1-3. The influence of glial cells on the accumulation and clearance of ß-amyloid (Aß) and neurofibrillary tau in the brains of individuals with Alzheimer's disease (AD) is poorly understood, despite growing awareness that these are therapeutically important interactions4,5. Here we show, in humans and mice, that astrocyte-sourced interleukin-3 (IL-3) programs microglia to ameliorate the pathology of AD. Upon recognition of Aß deposits, microglia increase their expression of IL-3Rα-the specific receptor for IL-3 (also known as CD123)-making them responsive to IL-3. Astrocytes constitutively produce IL-3, which elicits transcriptional, morphological, and functional programming of microglia to endow them with an acute immune response program, enhanced motility, and the capacity to cluster and clear aggregates of Aß and tau. These changes restrict AD pathology and cognitive decline. Our findings identify IL-3 as a key mediator of astrocyte-microglia cross-talk and a node for therapeutic intervention in AD.

NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism.

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.

Gene Editing for Corneal Stromal Regeneration.

CRISPR/Cas9 gene editing holds the promise of sequence-specific alteration of the genome to achieve therapeutic benefit in the treated tissue. Cas9 is an RNA-guided nuclease in which the sequence of the RNA can be altered to match the desired target. However, care must be taken in target choice and RNA guide design to ensure both maximum on-target and minimum off-target activity. The cornea is an ideal tissue for gene therapy due to its small surface area, accessibility, immune privilege, avascularity, and ease of visualization. Herein, we describe the design, testing, and delivery of Cas9 and guide RNAs to target genes expressed in the cornea.

Protein Analysis of the TGFBIR124H Mouse Model Gives Insight into Phenotype Development of Granular Corneal Dystrophy.

PURPOSE: Mutations in the transforming growth factor ß-induced protein (TGFBIp) are associated with TGFBI-linked corneal dystrophies, which manifests as protein deposits in the cornea. A total of 70 different disease-causing mutations have been reported so far including the common R124H substitution, which is associated with granular corneal dystrophy type 2 (GCD2). The disease mechanism of GCD2 is not known and the current treatments only offer temporary relief due to the reoccurrence of deposits. EXPERIMENTAL DESIGN: The corneal protein profiles of the three genotypes (wild-type (WT), heterozygotes, and homozygotes) of a GCD2 mouse model are compared using label-free quantitative LC-MS/MS. RESULTS: The mice do not display corneal protein deposits and the global protein expression between the three genotypes is highly similar. However, the expression of mutated TGFBIp is 41% of that of the WT protein. CONCLUSIONS AND CLINICAL RELEVANCE: It is proposed that the lowered expression level of mutant TGFBIp protein relative to WT protein is the direct cause of the missing development of corneal deposits in the mouse. The overall protein profiles of the corneas are not impacted by the reduced amount of TGFBIp. Altogether, this supports a partial reduction in mutated TGFBIp as a potential treatment strategy for GCD2.

Mutation-Independent Allele-Specific Editing by CRISPR-Cas9, a Novel Approach to Treat Autosomal Dominant Disease.

CRISPR-Cas9 provides a tool to treat autosomal dominant disease by non-homologous end joining (NHEJ) gene disruption of the mutant allele. In order to discriminate between wild-type and mutant alleles, Streptococcus pyogenes Cas9 (SpCas9) must be able to detect a single nucleotide change. Allele-specific editing can be achieved by using either a guide-specific approach, in which the missense mutation is found within the guide sequence, or a protospacer-adjacent motif (PAM)-specific approach, in which the missense mutation generates a novel PAM. While both approaches have been shown to offer allele specificity in certain contexts, in cases where numerous missense mutations are associated with a particular disease, such as TGFBI (transforming growth factor ß-induced) corneal dystrophies, it is neither possible nor realistic to target each mutation individually. In this study, we demonstrate allele-specific CRISPR gene editing independent of the disease-causing mutation that is capable of achieving complete allele discrimination, and we propose it as a targeting approach for autosomal dominant disease. Our approach utilizes natural variants in the target region that contain a PAM on one allele that lies in cis with the causative mutation, removing the constraints of a mutation-dependent approach. Our innovative patient-specific guide design approach takes into account the patient's individual genetic make-up, allowing on- and off-target activity to be assessed in a personalized manner.

Author Correction: Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Listeria Phages Induce Cas9 Degradation to Protect Lysogenic Genomes.

Bacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy phage (viral) DNA. Phages, in turn, have evolved diverse "anti-CRISPR" proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode two to three distinct anti-Cas9 proteins, with acrIIA1 always present. However, the significance of AcrIIA1's pervasiveness and its mechanism are unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain. During lysogeny in Listeria, AcrIIA1 triggers Cas9 degradation. During lytic infection, however, AcrIIA1 fails to block Cas9 due to its multi-step inactivation mechanism. Thus, phages encode an additional Acr that rapidly binds and inactivates Cas9. AcrIIA1 also uniquely inhibits a highly diverged Cas9 found in Listeria (similar to SauCas9) and Type II-C Cas9s, likely due to Cas9 HNH domain conservation. In summary, Listeria phages inactivate Cas9 in lytic growth using variable, narrow-spectrum inhibitors, while the broad-spectrum AcrIIA1 stimulates Cas9 degradation for protection of the lysogenic genome.

Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.

Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.

Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer.

CRISPR-Cas adaptive immune systems protect bacteria and archaea against their invading genetic parasites, including bacteriophages/viruses and plasmids. In response to this immunity, many phages have anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas targeting. To date, anti-CRISPR genes have primarily been discovered in phage or prophage genomes. Here, we uncovered acr loci on plasmids and other conjugative elements present in Firmicutes using the Listeria acrIIA1 gene as a marker. The four identified genes, found in Listeria, Enterococcus, Streptococcus and Staphylococcus genomes, can inhibit type II-A SpyCas9 or SauCas9, and are thus named acrIIA16-19. In Enterococcus faecalis, conjugation of a Cas9-targeted plasmid was enhanced by anti-CRISPRs derived from Enterococcus conjugative elements, highlighting a role for Acrs in the dissemination of plasmids. Reciprocal co-immunoprecipitation showed that each Acr protein interacts with Cas9, and Cas9-Acr complexes were unable to cleave DNA. Northern blotting suggests that these anti-CRISPRs manipulate single guide RNA length, loading or stability. Mirroring their activity in bacteria, AcrIIA16 and AcrIIA17 provide robust and highly potent broad-spectrum inhibition of distinct Cas9 proteins in human cells (for example, SpyCas9, SauCas9, SthCas9, NmeCas9 and CjeCas9). This work presents a focused analysis of non-phage Acr proteins, demonstrating a role in horizontal gene transfer bolstered by broad-spectrum CRISPR-Cas9 inhibition.

Effective In Vivo Topical Delivery of siRNA and Gene Silencing in Intact Corneal Epithelium Using a Modified Cell-Penetrating Peptide.

Autosomal dominantly inherited genetic disorders such as corneal dystrophies are amenable to allele-specific gene silencing with small interfering RNA (siRNA). siRNA delivered to the cornea by injection, although effective, is not suitable for a frequent long-term treatment regimen, whereas topical delivery of siRNA to the cornea is hampered by the eye surface's protective mechanisms. Herein we describe an attractive and innovative alternative for topical application using cell-penetrating peptide derivatives capable of complexing siRNA non-covalently and delivering them into the cornea. Through a rational design approach, we modified derivatives of a cell-penetrating peptide, peptide for ocular delivery (POD), already proved to diffuse into the corneal layers. These POD derivatives were able to form siRNA-peptide complexes (polyplexes) of size and ζ-potential similar to those reported able to undergo cellular internalization. Successful cytoplasmic release and gene silencing in vitro was obtained when an endosomal disruptor, chloroquine, was added. A palmitoylated-POD, displaying the best delivery properties, was covalently functionalized with trifluoromethylquinoline, an analog of chloroquine. This modified POD, named trifluoromethylquinoline-palmitoyl-POD (QN-Palm-POD), when complexed with siRNA and topically applied to the eye in vivo, resulted in up to 30% knockdown of luciferase reporter gene expression in the corneal epithelium. The methods developed within represent a valid standardized approach that is ideal for screening of a range of delivery formulations.

Personalised genome editing - The future for corneal dystrophies.

The potential of personalised genome editing reaching the clinic has come to light due to advancements in the field of gene editing, namely the development of CRISPR/Cas9. The different mechanisms of repair used to resolve the double strand breaks (DSBs) mediated by Cas9 allow targeting of a wide range of disease causing mutations. Collectively, the corneal dystrophies offer an ideal platform for personalised genome editing; the majority of corneal dystrophies are monogenic, highly penetrant diseases with a known pattern of inheritance. This genetic background coupled with the accessibility, ease of visualisation and immune privilege status of the cornea make a gene editing strategy for the treatment of corneal dystrophies an attractive option. Off-target cleavage is a major concern for the therapeutic use of CRISPR/Cas9, thus current efforts in the gene editing field are focused on improving the genome-wide specificity of Cas9 to minimise the risk of off-target events. In addition, the delivery of CRISPR/Cas9 to different tissues is a key focus; various viral and non-viral platforms are being explored to develop a vehicle that is highly efficient, specific and non-toxic. The rapid pace and enthusiasm with which CRISPR/Cas9 has taken over biomedical research has ensured the personalised medicine revolution has been realised. CRISPR/Cas9 has recently been utilised in the first wave of clinical trials, and the potential for a genome editing therapy to treat corneal dystrophies looks promising. This review will discuss the current status of therapeutic gene editing in relation to the corneal dystrophies.

Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders.

CRISPR/Cas9 holds immense potential to treat a range of genetic disorders. Allele-specific gene disruption induced by non-homologous end-joining (NHEJ) DNA repair offers a potential treatment option for autosomal dominant disease. Here, we successfully delivered a plasmid encoding S. pyogenes Cas9 and sgRNA to the corneal epithelium by intrastromal injection and acheived long-term knockdown of a corneal epithelial reporter gene, demonstrating gene disruption via NHEJ in vivo. In addition, we used TGFBI corneal dystrophies as a model of autosomal dominant disease to assess the use of CRISPR/Cas9 in two allele-specific systems, comparing cleavage using a SNP-derived PAM to a guide specific approach. In vitro, cleavage via a SNP-derived PAM was found to confer stringent allele-specific cleavage, while a guide-specific approach lacked the ability to distinguish between the wild-type and mutant alleles. The failings of the guide-specific approach highlights the necessity for meticulous guide design and assessment, as various degrees of allele-specificity are achieved depending on the guide sequence employed. A major concern for the use of CRISPR/Cas9 is its tendency to cleave DNA non-specifically at "off-target" sites. Confirmation that S. pyogenes Cas9 lacks the specificity to discriminate between alleles differing by a single base-pair regardless of the position in the guide is demonstrated.