Fig. 3: An inducible DNA repair CRISPRa/i system for enhancing HDR efficiency.

a Diagram of TRE-MPH and TRE-CK expression vectors used to activate CDK1 and repress KU80, respectively. rtTA interact with doxycycline, the complex could bind to TRE3G promoter, which then initiate the transcription of MCP-P65-HSF1 or COM-KRAB. b The workflow of establishing inducible HDR increasing system. Activation of CDK1 and/or repression of KU80 can be achieved by simply controlling the availability of doxycycline. Dox, doxycycline; Puro, puromycin. c-e HEK293-TRE-MPH, HEK293-TRE-CK, and HEK293-TRE-MPH-CK cell lines were obtained based on HEK293-Cas9 cell line by G418 selection. Several random clones were picked for each cell line. Although the transcriptional levels of CDK1 activation or KU80 repression can vary between clones, the clones with significant CDK1 activation and/or KU80 repression have increased HDR efficiency. The transcription level of CDK1 and KU80 were determined by RT-qPCR after 2 days of doxycycline treatment. f Quantitative analysis result of HDR efficiency (HEK293-TRE-MPH vs. HEK293-TRE-MPH + Dox, p = 0.001; HEK293-TRE-CK vs. HEK293-TRE-CK + Dox, p = 0.0011; HEK293-TRE-MPH-CK vs. HEK293-TRE-MPH-CK + Dox, p = 0.0032). Data was shown as the mean ± SEM from three independent experiments. Significance was calculated using the Paired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001