CRISPR/Cas9-Based Cellular Engineering for Targeted Gene Overexpression

Abstract

Gene and cellular therapies hold tremendous promise as agents for treating genetic disorders. However, the effective delivery of genes, particularly large ones, and expression at therapeutic levels can be challenging in cells of clinical relevance. To address this engineering hurdle, we sought to employ the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to insert powerful regulatory elements upstream of an endogenous gene. We achieved robust activation of the COL7A1 gene in primary human umbilical cord blood CD34+ hematopoietic stem cells and peripheral blood T-cells. CD34+ cells retained their colony forming potential and, in a second engineering step, we disrupted the T-cell receptor complex in T-cells. These cellular populations are of high translational impact due to their engraftment potential, broad circulatory properties, and favorable immune profile that supports delivery to multiple recipients. This study demonstrates the feasibility of targeted knock in of a ubiquitous chromatin opening element, promoter, and marker gene that doubles as a suicide gene for precision gene activation. This system merges the specificity of gene editing with the high level, sustained gene expression achieved with gene therapy vectors. We predict that this design concept will be highly transferrable to most genes in multiple model systems representing a facile cellular engineering platform for promoting gene expression.

Gene Therapy for Epidermolysis Bullosa

“…These results stimulated exploration of nonviral gene transfer methods including transposon, integrase (Ortiz-Urda et al, 2003b, 2003c, and adeno-associated virus . Clustered regularly interspaced short palindromic repeats (i.e., CRISPR)/Cas9-and transcription activator-like effector nuclease (TALEN)-based gene correction methodologies have also become active areas of exploration in EB therapy (Osborn et al, 2013(Osborn et al, , 2018Webber et al, 2016). Despite these new areas of study, ex vivo retroviral therapy has remained of high interest because of the efficiency of gene transfer to primary cells.…”

Molecular Characterization of Paralichthys olivaceus MAF1 and Its Potential Role as an Anti-Viral Hemorrhagic Septicaemia Virus Factor in Hirame Natural Embryo Cells

“…The CRISPR/Cas9 system enables genome editing and has revolutionized genetic improvement of fish, so we can apply it to upregulate PoMaf1 . To enhance target gene expression, regulatory elements are inserted upstream of an endogenous gene by a CRISPR/Cas9-mediated knock-in strategy [ 46 ]. In contrast, cis -regulatory elements (CREs) capable of silencing a gene promoter region can be targeted by the CRISPR/Cas9 system without adding exogenous genes [ 47 ].…”

Context-Dependent Strategies for Enhanced Genome Editing of Genodermatoses

“…However, as these approaches rely on the introduction of insertions and deletions within target loci, they are unsuitable for many mutations and diseases. It is primarily for this reason that the earliest applications of genome editing in genodermatoses typically involved exon/gene insertions [35,[40][41][42][43][44][45][46]. These strategies primarily involved the HR-based replacement of pathogenic exons with a non-pathogenic sequence encoding intronic selection markers, enabling simpler isolation of corrected cells, from early-stage and therefore inefficient approaches.…”

“…Barely detectable 0.48% correct HR efficiency made positive selection mandatory [45]. Osborn et al [46] employed a different strategy, aiming for distinct cell types as a source of missing C7 expression in RDEB. For their strategy a sgRNA targeting 164 bp upstream of the COL7A1 start codon and a donor template carrying a transcriptional promoting element termed UMET, flanked by donor arms spanning the COL7A1 transcriptional start site, were designed.…”

“…Therefore, the donor template was packaged into AAV-6 serotype and cluster of differentiation 34 (CD34) + CD133 + HSCs were infected along with Cas9 RNP complexes electroporated. This efficient gene targeting and HR inducing method (>60%) allowed high C7 levels in HSCs and T-cells [46].…”