Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain

Abstract

Genome-editing technology has revolutionized the field of biology. Here, we report a novel de novo gene-targeting method mediated by in utero electroporation into the developing mammalian brain. Electroporation of donor DNA with the CRISPR/Cas9 system vectors successfully leads to knock-in of the donor sequence, such as EGFP, to the target site via the homology-directed repair mechanism. We developed a targeting vector system optimized to prevent anomalous leaky expression of the donor gene from the plasmid, which otherwise often occurs depending on the donor sequence. The knock-in efficiency of the electroporated progenitors reached up to 40% in the early stage and 20% in the late stage of the developing mouse brain. Furthermore, we inserted different fluorescent markers into the target gene in each homologous chromosome, successfully distinguishing homozygous knock-in cells by color. We also applied this de novo gene targeting to the ferret model for the study of complex mammalian brains. Our results demonstrate that this technique is widely applicable for monitoring gene expression, visualizing protein localization, lineage analysis and gene knockout, all at the single-cell level, in developmental tissues.

Trisomy silencing by XIST normalizes Down syndrome cell pathogenesis demonstrated for hematopoietic defects in vitro

“…Additionally, bone marrow transplantation of genetically modified HSC is actively pursued for clinical applications and cord blood could serve as a more accessible source of HSCs for all DS newborns. Since it has been shown in mice that Xist can initiate chromosome silencing specifically in somatic hematopoietic progenitor cells 46 , it can now be considered that dosage-compensation of chromosome 21 expression in DS-TMD children might eventually be developed as a therapeutic ex vivo or in vivo strategy, or even conceivably in fetal liver in utero 47 , 48 (the origin of cells that give rise to TMD and leukemia). It is also important to note that the blood system has relevance to other bodily systems impacted by trisomy 21.…”

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

“…In these experiments, viral vectors, such as AAVs, lentiviruses, and adenoviruses have been involved in infecting the fetuses at days 9-15 of pregnancy. Experiments using in utero gene delivery of genome-editing components have been performed by several research groups [17,[91][92][93][94]. Ricciardi et al [93] demonstrated that intra-amniotic administration of polymeric, biodegradable nanoparticles derived from poly (lactic-co-glycolic acid) containing triplex-forming peptide nucleic acids and donor ssDNAs into mouse fetuses carrying mutations in the β-globin gene, results in the offspring being rescued from a disease similar to human β-thalassemia.…”

“…No recipients are required [91][92][93][94] Abbreviations: AAV, adeno-associated virus; EP, electroporation; GONAD, Genome-editing via oviductal nucleic acids delivery; i-GONAD, improved GONAD; ICSI, intracytoplasmic injection of sperm; TPGD, transplacental gene delivery; TPGD-GEF, transplacental gene delivery for acquiring genome-edited fetuses.…”

Biophysics of Biochemical Signaling in Dendritic Spines: Implications in Synaptic Plasticity

“…Both of these would slow down the kinetics of the small GTPase activity. Recent advances in techniques for labeling endogenous proteins in specific cells including in vivo genome editing (67)(68)(69)(70), conditional knock-in (71), and intrabody (72) will open the possibility of imaging the activity of endogenous proteins. Thus, we expect to be able to image interactions and conformation changes of endogenous proteins using this approach.…”