Mackaylanier1438

Studies of knockout (KO) mice with defects in the endolysosomal two-pore channels (TPCs) have shown TPCs to be involved in pathophysiological processes, including heart and muscle function, metabolism, immunity, cancer, and viral infection. With the objective of studying TPC2's pathophysiological roles for the first time in a large, more humanlike animal model, TPC2 KO pigs were produced using CRISPR-Cas9. A major problem using CRISPR-Cas9 to edit embryos is mosaicism; thus, we studied for the first time the effect of microinjection timing on mosaicism. Mosaicism was greatly reduced when in vitro produced embryos were microinjected before insemination, and surgical embryo transfer (ET) was performed using such embryos. All TPC2 KO fetuses and piglets born following ET (i.e., F0 generation) were nonmosaic biallelic KOs. The generation of nonmosaic animals greatly facilitates germ line transmission of the mutation, thereby aiding the rapid and efficient generation of KO animal lines for medical research and agriculture.Genome editing using CRISPR-Cas9 has produced a functional cure for a small number of patients with sickle cell disease and beta-thalassemia. Rather than repairing the causative mutation, this striking outcome was attained by the knockout of a lineage-specific regulatory element for a gene, BCL11A, that controls fetal hemoglobin levels a first example of clinical success in targeting a locus initially identified in a genome-wide association study, and formal proof of the "in the age of CRISPR, the entire genome is a druggable target" notion. This remarkable development, along with advancement to the clinic of several additional editing-based approaches to the hemoglobinopathies, highlights a sense of urgency in accelerating scientific, regulatory, and public health innovation that will allow broad and equitable access to editing-based cures.Conventional CRISPR approaches for precision genome editing rely on the introduction of DNA double-strand breaks (DSB) and activation of homology-directed repair (HDR), which is inherently genotoxic and inefficient in somatic cells. The development of base editing (BE) systems that edit a target base without requiring generation of DSB or HDR offers an alternative. Here, we describe a novel BE system called Pin-pointTM that recruits a DNA base-modifying enzyme through an RNA aptamer within the gRNA molecule. Pin-point is capable of efficiently modifying base pairs in the human genome with precision and low on-target indel formation. This system can potentially be applied for correcting pathogenic mutations, installing premature stop codons in pathological genes, and introducing other types of genetic changes for basic research and therapeutic development.In complex multicellular systems, gene expression is regulated at multiple stages through interconnected complex molecular pathways and regulatory networks. Transcription is the first step in gene expression and is subject to multiple layers of regulation in which epigenetic mechanisms such as DNA methylation, histone tail modifications, and chromosomal conformation play an essential role. In recent years, CRISPR-Cas9 systems have been employed to unearth this complexity and provide new insights on the contribution of chromatin dysregulation in the development of genetic diseases, as well as new tools to prevent or reverse this dysregulation. In this review, we outline the recent development of a variety of CRISPR-based epigenetic editors for targeted DNA methylation/demethylation, histone modification, and three-dimensional DNA conformational change, highlighting their relative performance and impact on gene regulation. Finally, we provide insights on the future developments aimed to accelerate our understanding of the causal relationship between epigenetic marks, genome organization, and gene regulation.Since observations that CRISPR nucleases function in mammalian cells, many strategies have been devised to adapt them for genetic engineering. Here, we investigated self-cutting and integrating CRISPR-Cas9 plasmids (SCIPs) as easy-to-use gene editing tools that insert themselves at CRISPR-guided locations. SCIPs demonstrated similar expression kinetics and gene disruption efficiency in mouse (EL4) and human (Jurkat) cells, with stable integration in 3-6% of transfected cells. Clonal sequencing analysis indicated that integrants showed bi- or mono-allelic integration of entire CRISPR plasmids in predictable orientations and with limited insertion or deletion formation. Interestingly, including longer homology arms (HAs; 500 bp) in varying orientations only modestly increased knock-in efficiency (by around twofold). fMLP Using a SCIP-payload design (SCIPpay) that liberates a promoter-less sequence flanked by HAs thereby requiring perfect homology-directed repair for transgene expression, longer HAs resulted in higher integration efficiency and precision of the payload but did not affect integration of the remaining plasmid sequence. As proofs of concept, we used SCIPpay to insert (1) a gene fragment encoding tdTomato into the CD69 locus of Jurkat cells, thereby creating a cell line that reports T-cell activation, and (2) a chimeric antigen receptor gene into the TRAC locus. Here, we demonstrate that SCIPs function as simple, efficient, and programmable tools useful for generating gene knock-out/knock-in cell lines, and we suggest future utility in knock-in site screening/optimization, unbiased off-target site identification, and multiplexed, iterative, and/or library-scale automated genome engineering.Adenine base editors (ABEs) can correct gene mutations without creating double-strand breaks. However, in recent reports, these editors showed guide-independent RNA off-target activities. This work describes our development of a delivery method to minimize ABEs' RNA off-target activity. After discovering a RNA off-target hot spot for sensitive detection of RNA off-target activities, we found that delivering ribonucleoproteins (RNPs) by electroporation generated undetectable non-specific RNA editing, but on-target base editing activity was also relatively low. We then explored a lentivirus capsid-based delivery strategy to deliver ABE. We used aptamer/aptamer-binding protein (ABP) interactions to package ABE RNPs into lentiviral capsids. Capsid RNPs were delivered to human cells for highly efficient guided base editing. Importantly, RNA off-target activities from the capsid RNPs were undetectable. Our new lentiviral capsid-based ABE RNP delivery method with minimal RNA off-target activities makes ABE one step closer to possible therapeutic applications.