Nanoparticle-mediated genome editing in single-cell embryos via peptide nucleic acids.

Through preimplantation genetic diagnosis, genetic diseases can be detected during the early stages of embryogenesis, but effective treatments for many of these disorders are lacking. Gene editing could allow for correction of the underlying mutation during embryogenesis to prevent disease pathogenesis or even provide a cure. Here, we demonstrate that administration of peptide nucleic acids and single-stranded donor DNA oligonucleotides encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles to single-cell embryos allows for editing of an eGFP-beta globin fusion transgene. Blastocysts from treated embryos exhibit high levels of editing (~94%), normal physiological development, normal morphology, and no detected off-target genomic effects. Treated embryos reimplanted to surrogate moms show normal growth without gross developmental abnormalities and with no identified off-target effects. Mice from reimplanted embryos consistently show editing, characterized by mosaicism across multiple organs with some organ biopsies showing up to 100% editing. This proof-of-concept work demonstrates for the first time the use of peptide nucleic acid (PNA)/DNA nanoparticles as a means to achieve embryonic gene editing.

A Human MSH6 Germline Variant Associated With Systemic Lupus Erythematosus Induces Lupus-like Disease in Mice.

OBJECTIVE: To determine if single-nucleotide polymorphisms (SNPs) in DNA repair genes are enriched in individuals with systemic lupus erythematosus (SLE) and if they are sufficient to confer a disease phenotype in a mouse model. METHODS: Human exome chip data of 2499 patients with SLE and 1230 healthy controls were analyzed to determine if variants in 10 different mismatch repair genes (MSH4, EXO1, MSH2, MSH6, MLH1, MSH3, POLH, PMS2, ML3, and APEX2) were enriched in individuals with SLE. A mouse model of the MSH6 SNP, which was found to be enriched in individuals with SLE, was created using CRISPR/Cas9 gene targeting. Wildtype mice and mice heterozygous and homozygous for the MSH6 variant were then monitored for 2 years for the development of autoimmune phenotypes, including the presence of high levels of antinuclear antibodies (ANA). Additionally, somatic hypermutation frequencies and spectra of the intronic region downstream of the VH J558-rearranged JH4 immunoglobulin gene was characterized from Peyer's patches. RESULTS: Based on the human exome chip data, the MSH6 variant (rs63750897, p.Ser503Cys) is enriched among patients with SLE versus controls after we corrected for ancestry (odds ratio = 8.39, P = 0.0398). Mice homozygous for the MSH6 variant (Msh6S502C/S502C ) harbor significantly increased levels of ANA. Additionally, the Msh6S502C/S502C mice display a significant increase in the infiltration of CD68+ cells (a marker for monocytes and macrophages) into the lung alveolar space as well as apoptotic cells. Furthermore, characterization of somatic hypermutation in these mice reveals an increase in the DNA polymerase η mutational signature. CONCLUSION: An MSH6 mutation that is enriched in humans diagnosed with lupus was identified. Mice harboring this Msh6 mutation develop increased autoantibodies and an inflammatory lung disease. These results suggest that the human MSH6 variant is linked to the development of SLE.

The hematopoietic compartment is sufficient for lupus development resulting from the POLB-Y265C mutation.

Systemic lupus erythematosus is a chronic disease characterized by autoantibodies, renal and cutaneous disease, and immune complex formation. Emerging evidence suggests that aberrant DNA repair is an underlying mechanism of lupus development. We previously showed that the POLBY265C/C mutation, which results in development of an aberrant immune repertoire, leads to lupus-like disease in mice. To address whether the hematopoietic compartment is sufficient for lupus development, we transplanted bone marrow cells from POLBY265C/C and POLB+/+ into wild-type congenic mice. Only mice transplanted with the POLBY265C/C bone marrow develop high levels of antinuclear antibodies and renal disease. In conclusion, we show that the hematopoietic compartment harvested from the POLBY265C/C mice is sufficient for development of autoimmune disease.

Antispacer peptide nucleic acids for sequence-specific CRISPR-Cas9 modulation.

Despite the rapid and broad implementation of CRISPR-Cas9-based technologies, convenient tools to modulate dose, timing, and precision remain limited. Building on methods using synthetic peptide nucleic acids (PNAs) to bind RNA with unusually high affinity, we describe guide RNA (gRNA) spacer-targeted, or 'antispacer', PNAs as a tool to modulate Cas9 binding and activity in cells in a sequence-specific manner. We demonstrate that PNAs rapidly and efficiently target complexed gRNA spacer sequences at low doses and without design restriction for sequence-selective Cas9 inhibition. We further show that short PAM-proximal antispacer PNAs achieve potent cleavage inhibition (over 2000-fold reduction) and that PAM-distal PNAs modify gRNA affinity to promote on-target specificity. Finally, we apply antispacer PNAs for temporal regulation of two dCas9-fusion systems. These results present a novel rational approach to nucleoprotein engineering and describe a rapidly implementable antisense platform for CRISPR-Cas9 modulation to improve spatiotemporal versatility and safety across applications.

Peptide nucleic acids and their role in gene regulation and editing.

The unique properties of peptide nucleic acid (PNA) makes it a desirable candidate to be used in therapeutic and biotechnological interventions. It has been broadly utilized for numerous applications, with a major focus in regulation of gene expression, and more recently in gene editing. While the classic PNA design has mainly been employed to date, chemical modifications of the PNA backbone and nucleobases provide an avenue to advance the technology further. This review aims to discuss the recent developments in PNA based gene manipulation techniques and the use of novel chemical modifications to improve the current state of PNA mediated gene targeting.