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.

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 CD44CD4 T cells generated interleukin-3 (IL-3), while microglia and recruited myeloid cells expressed interleukin-3 receptor-ɑ (IL-3Rɑ).

Base editing as a genetic treatment for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the gene. Despite the development of various therapies, outcomes can remain suboptimal in SMA infants and the duration of such therapies are uncertain. is a paralogous gene that mainly differs from by a C•G-to-T•A transition in exon 7, resulting in the skipping of exon 7 in most transcripts and production of only low levels of survival motor neuron (SMN) protein.

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.

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 health. The influence of glial cells on the accumulation and clearance of β-amyloid (Aβ) and neurofibrillary tau in the brains of individuals with Alzheimer's disease (AD) is poorly understood, despite growing awareness that these are therapeutically important interactions. Here we show, in humans and mice, that astrocyte-sourced interleukin-3 (IL-3) programs microglia to ameliorate the pathology of 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.

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.

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.

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.

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.

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 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).

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.

Evaluation of TGFBI corneal dystrophy and molecular diagnostic testing.

To date, 70 different TGFBI mutations that cause epithelial-stromal corneal dystrophies have been described. At present one commercially available test examines for the five most common of these mutations: R124H, R124C, R124L, R555W, and R555Q. To expand the capability of identifying the causative mutation in the remaining cases, 57 mutations would need to be added.

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.

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.