Efficient gene editing through an intronic selection marker in cells

Abstract

Background Gene editing technology has provided researchers with the ability to modify genome sequences in almost all eukaryotes. Gene-edited cell lines are being used with increasing frequency in both bench research and targeted therapy. However, despite the great importance and universality of gene editing, the efficiency of homology-directed DNA repair (HDR) is too low, and base editors (BEs) cannot accomplish desired indel editing tasks. Results and discussion Our group has improved HDR gene editing technology to indicate DNA variation with an independent selection marker using an HDR strategy, which we named Gene Editing through an Intronic Selection marker (GEIS). GEIS uses a simple process to avoid nonhomologous end joining (NHEJ)-mediated false-positive effects and achieves a DsRed positive rate as high as 87.5% after two rounds of fluorescence-activated cell sorter (FACS) selection without disturbing endogenous gene splicing and expression. We re-examined the correlation of the conversion tract and efficiency, and our data suggest that GEIS has the potential to edit approximately 97% of gene editing targets in human and mouse cells. The results of further comprehensive analysis suggest that the strategy may be useful for introducing multiple DNA variations in cells.

TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage

“…Targeting introns in nuclease assisted gene editing approaches has the advantage of minimizing deleterious effects that indels might have at the targeted region. Indeed, editing intronic regions to express a transgene of interest has been reported in multiple studies 23,24,25,26,27 . However, we repurposed this strategy by using a promoter-less transgene that hijacks the regulatory element of the myeloid specific CD11b gene without affecting its endogenous expression.…”

TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage

“…Targeting introns in nuclease assisted gene editing approaches has the advantage of minimizing deleterious effects that indels might have at the targeted region. Indeed, editing intronic regions to express a transgene of interest has been reported in multiple studies 23,24,25,26,27 .…”

Disrupting the phase separation of KAT8–IRF1 diminishes PD-L1 expression and promotes antitumor immunity

“…The knock-in experiment was performed as previously described 63 . An efficient IRF1 -targeting sgRNA was selected, and a DNA donor template containing mutations was designed.…”