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Fig. 3 | Molecular Cancer

Fig. 3

From: Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing

Fig. 3

Applications of Cas9 variants. a Double-stranded DNA breaks (DSBs) generated by the Cas9 nuclease will be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) within the cell. During NHEJ, the ligase frequently adds random insertions and deletions (indels) to repair the break site. This process is considered asĀ error-prone and used to cause generalized gene disruptions, typically with a loss-of-function outcome. HDR uses a repair template that has homology with sites upstream and downstream of the cut site, allowing for recombination of the template for an error-free repair. For gene editing purposes, an exogenous donor DNA repair template can be designed to insert large foreign DNA constructs into the site, allowing for site-specific knock-in that can result in reduction or gain of activity at the desired locus, or replacement with a new gene cassette. b nCas9 can be tethered to cytidine and adenosine deaminases to allow for single-base pair editing. Cytidine deaminase catalyzes the conversion of CT while adenosine deaminase will catalyze the conversion of AG. Base editing can be used to cause gene disruption without generating DSBs by altering coding regions to introduce premature STOP codons or to interfere with splicing donor/acceptor sites. nCas9 can also be fused to reverse transcriptase (RT), together with a specialized guide RNA (pegRNA) forming a complex that can be used to introduce larger sequence additions into a DNA region without creating DSBs. c Enzymatically dead Cas9 (dCas9) can be tethered to transcriptional activators (e.g., VP64) to potentiate transcription at a specific promotor region targeted by the gRNA, while transcriptional repressors (e.g., KRAB) can attenuate transcriptional activity. dCas9 can be tethered to epigenetic modifying enzymes to catalyze demethylation (e.g., TETs) or active methylation (DNMTs) for temporal regulation of epigenetic motifs and transcriptional activity at gRNA-specified loci promoters. Created with BioRender.com

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