![]() Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene, and bypasses the need to work with clones. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knock-out/rescue in the closely related Rab11 family of vesicle trafficking regulators. These approaches combine blockade of active gene expression with the ability to re-activate expression on demand, including activation of silenced genes. Here, we present DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon combined with auxin-mediated targeted protein degradation), and LUXon (light responsive DExCon) approaches which combine one-step CRISPR-Cas9-mediated targeted knockin of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. However, once inactivated, their re-activation remains difficult, especially in diploid cells. This review outlines the current developments in optimization of templates for the GE mediated therapeutic gene correction.ĬRISPR technology has made generation of gene knock-outs widely achievable in cells. Furthermore, tuning of editing conditions for the chosen template as well as its structure, length, sequence optimization, homology arm (HA) modifications may have paramount importance for achieving highly efficient knock-in with favorable safety profile. While viral vectors with notable example of AAVs as a donor template carrier remain the mainstay in many preclinical trials, non-viral templates, including plasmid and linear dsDNA, long ssDNA templates, single and double-stranded ODNs, represent a promising alternative. However, emerging data suggests that optimal characteristics of repair templates may play an important role in the knock-in mechanisms. Significant efforts were focused on improving the parameters and interaction of GE nuclease proteins. Currently, the efficiency of methods for GE-mediated knock-in is limited. By introducing a double strand break (DSB) in the defined locus of genomic DNA, GE tools allow to knockout the desired gene or to knock-in the therapeutic gene if provided with an appropriate repair template. Genome editing (GE) now is an increasingly important technology for achieving stable therapeutic effect in gene correction, with hematopoietic cells representing a key target cell population for developing novel treatments for a number of hereditary diseases, infections and cancer. Gene therapy is a fast developing field of medicine with hundreds of ongoing early-stage clinical trials and numerous preclinical studies. ![]() Example of mis-integration identified from cells engineered using plasmid donor: integration of donor plasmid backbone sequence. Examples of mis-integration identified from cells engineered using ssDNA donor: truncated insertion of GFP payload. Examples of mis-integration identified from cells engineered using PCR donor: blunt insertion resulting in duplication of the homology arms, and integration of donor concatemer. Sequence alignment mismatches between sequencing read and reference sequences are indicated: cross for sequence mismatch, down-tick for 1-bp insertions, up-tick for deletions. Final results are available within an interactive visualization framework including relevant sequence boundaries (forward and reverse sequencing primers, homology arms and GFP payload) are highlighted in colors. The architecture of each read is further analyzed and classified into phenotypic outcomes. Each sequencing read (black) is deconstructed through a series of local alignments to either the reference genome (blue) or any other sequence provided, in particular the donor template (orange). Lengths corresponding to wt + in-dels and expected HDR product are shaded in grey. (Right) Corresponding distribution frequency of amplicon length after selection of GFP+ cells and SMRT long-read amplicon sequencing of RAB11A. (Left) Flow-cytometry fluorescence profiles of GFP insertion at the RAB11A N-terminus in HEK293t cells using plasmid, PCR or long ssDNA donors. Experimental design: HDR knock-in of GFP in open-reading frames (ORFs), followed by amplicon sequencing. Knock-knock analysis of repair architecture.
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