Challenging Safety and Efficacy of Retinal Gene Therapies by Retinogenesis

Gene-expression programs modulated by transcription factors (TFs) mediate key developmental events. Here, we show that the synthetic transcriptional repressor (TR; ZF6-DB), designed to treat Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RHO-adRP), does not perturb murine retinal development, while maintaining its ability to block Rho expression transcriptionally. To express ZF6-DB into the developing retina, we pursued two approaches, (i) the retinal delivery (somatic expression) of ZF6-DB by Adeno-associated virus (AAV) vector (AAV-ZF6-DB) gene transfer during retinogenesis and (ii) the generation of a transgenic mouse (germ-line transmission, TR-ZF6-DB). Somatic and transgenic expression of ZF6-DB during retinogenesis does not affect retinal function of wild-type mice. The P347S mouse model of RHO-adRP, subretinally injected with AAV-ZF6-DB, or crossed with TR-ZF6-DB or shows retinal morphological and functional recovery. We propose the use of developmental transitions as an effective mode to challenge the safety of retinal gene therapies operating at genome, transcriptional, and transcript levels.

Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricket Gryllus bimaculatus

Studies of traditional model organisms like the fruit fly Drosophila melanogaster have contributed immensely to our understanding of the genetic basis of developmental processes. However, the generalizability of these findings cannot be confirmed without functional genetic analyses in additional organisms. Direct genome editing using targeted nucleases has the potential to transform hitherto poorly-understood organisms into viable laboratory organisms for functional genetic study. To this end, here we present a method to induce targeted genome knock-out and knock-in of desired sequences in an insect that serves as an informative contrast to Drosophila, the cricket Gryllus bimaculatus. The efficiency of germ line transmission of induced mutations is comparable to that reported for other well-studied laboratory organisms, and knock-ins targeting introns yields viable, fertile animals in which knock-in events are directly detectable by visualization of a fluorescent marker in the expression pattern of the targeted gene. Combined with the recently assembled and annotated genome of this cricket, this knock-in/knock-out method increases the viability of G. bimaculatus as a tractable system for functional genetics in a basally branching insect.

Epigenetic Events and Germ Line Transmission in Rag1 knock-out Chimeric Mice Produced by CRISPR/Cas9 Technology

Considering the effects of epigenetic changes on the embryonic development through altering the gene expression of pluripotency, trophectoderm, and imprinting genes, we determined the pattern of epigenetic alterations in transgenic embryos produced by injecting transgenic mESCs into the morula. To aim this, RAG1 knock-out mESCs were produced using CRISPR-Cas9 editing system and then microinjected into morulas to develop chimeric embryos. Afterward, the expression of pluripotency, trophectoderm genes, and imprinting genes in these embryos were evaluated using real-time PCR. Immunohistochemical analysis was carried out to determine the methylation rates of H3K9me3 and H3K4me3. In the following, since epigenetic alterations can be at least one of the possible reasons of male infertility and the loss of germ-line transmission capacity, the germ-line transmission ability in chimeric mice was also evaluated by breeding them and subsequent backcrosses to wild type strains. Our findings showed that the methylation rates of H3K4me3 and H3K9me3 were significantly lower and higher respectively in the RAG1 knock-out embryos compared others groups. Moreover, the chimeric embryos exhibited the decreased expression of NANOG, OCT4, CDX2, TEAD4, and H19 genes. Following the breeding of chimeric males with normal female mouse, 40% of chimeras had no germ-line transmission (GLT), and 60% were infertile.We showed that the manipulation of mESC by CRISPR-Cas9 approach remarkably changed the methylation status of H3K9me3 and H3K4me3, resulting in impaired development of embryos through dysregulation of genes involved embryonic development and then, may be one of the reasons of infertility and lack of GLT.

Precise genome engineering inDrosophilausing prime editing

Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double-strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organismDrosophila melanogasterand developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely introduce premature stop codons in three classical visible marker genes,ebony,white, andforked. Furthermore, by restricting editing to germ cells, we demonstrate efficient germ-line transmission of a precise edit inebonyto 36% of progeny. Our results suggest that prime editing is a useful system inDrosophilato study gene function, such as engineering precise point mutations, deletions, or epitope tags.

Precise genome engineering in Drosophila using prime editing

Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organism Drosophila melanogaster and developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely installed premature stop codons in three classical visible marker genes, ebony, white, and forked. Furthermore, by restricting editing to germ cells, we demonstrate efficient germ line transmission of a precise edit in ebony to ~50% of progeny. Our results suggest that prime editing is a useful system in Drosophila to study gene function, such as engineering precise point mutations, deletions, or epitope tags.

Inherited chromosomally integrated human herpesvirus 6 as a predisposing risk factor for the development of angina pectoris

Inherited chromosomally integrated human herpesvirus-6 (iciHHV-6) results in the germ-line transmission of the HHV-6 genome. Every somatic cell of iciHHV-6+ individuals contains the HHV-6 genome integrated in the telomere of chromosomes. Whether having iciHHV-6 predisposes humans to diseases remains undefined. DNA from 19,597 participants between 40 and 69 years of age were analyzed by quantitative PCR (qPCR) for the presence of iciHHV-6. Telomere lengths were determined by qPCR. Medical records, hematological, biochemical, and anthropometric measurements and telomere lengths were compared between iciHHV-6+ and iciHHV-6− subjects. The prevalence of iciHHV-6 was 0.58%. Two-way ANOVA with a Holm–Bonferroni correction was used to determine the effects of iciHHV6, sex, and their interaction on continuous outcomes. Two-way logistic regression with a Holm–Bonferroni correction was used to determine the effects of iciHHV6, sex, and their interaction on disease prevalence. Of 50 diseases monitored, a single one, angina pectoris, is significantly elevated (3.3×) in iciHHV-6+ individuals relative to iciHHV-6− subjects (P = 0.017; 95% CI, 1.73–6.35). When adjusted for potential confounding factors (age, body mass index, percent body fat, and systolic blood pressure), the prevalence of angina remained three times greater in iciHHV-6+ subjects (P = 0.015; 95%CI, 1.23–7.15). Analyses of telomere lengths between iciHHV-6− without angina, iciHHV-6− with angina, and iciHHV-6+ with angina indicate that iciHHV-6+ with angina have shorter telomeres than age-matched iciHHV-6− subjects (P = 0.006). Our study represents, to our knowledge, the first large-scale analysis of disease association with iciHHV-6. Our results are consistent with iciHHV-6 representing a risk factor for the development of angina.

A selfish DNA element engages a meiosis-specific motor and telomeres for germ-line propagation

The chromosome-like mitotic stability of the yeast 2 micron plasmid is conferred by the plasmid proteins Rep1-Rep2 and the cis-acting locus STB, likely by promoting plasmid-chromosome association and segregation by hitchhiking. Our analysis reveals that stable plasmid segregation during meiosis requires the bouquet proteins Ndj1 and Csm4. Plasmid relocalization from the nuclear interior in mitotic cells to the periphery at or proximal to telomeres rises from early meiosis to pachytene. Analogous to chromosomes, the plasmid undergoes Csm4- and Ndj1-dependent rapid prophase movements with speeds comparable to those of telomeres. Lack of Ndj1 partially disrupts plasmid–telomere association without affecting plasmid colocalization with the telomere-binding protein Rap1. The plasmid appears to engage a meiosis-specific motor that orchestrates telomere-led chromosome movements for its telomere-associated segregation during meiosis I. This hitherto uncharacterized mode of germ-line transmission by a selfish genetic element signifies a mechanistic variation within the shared theme of chromosome-coupled plasmid segregation during mitosis and meiosis.