Genome editing tools targeting high-risk human papillomavirus (HPV) oncogene could be a promising therapeutic strategy for the treatment of HPV-related cervical cancer. We aimed to improve the editing efficiency and detect off-target effects concurrently for the clinical translation strategy by using CRISPR-Cas9 system co-transfected with 34nt non-homologous double-stranded oligodeoxynucleotide (dsODN). We firstly tested this strategy on targeting the Green Fluorescent Protein (GFP) gene, of which the expression is easily observed. Our results showed that the GFP+ cells were significantly decreased when using GFP-sgRNAs with dsODN, compared to using GFP-sgRNAs without donors. By PCR and Sanger sequencing, we verified the dsODN integration into the break sites of the GFP gene. And by amplicon sequencing, we observed that the indels% of the targeted site on the GFP gene was increased by using GFP-sgRNAs with dsODN. Next, we went on to target the HPV18 E7 oncogene by using single E7-sgRNA and multiplexed E7-sgRNAs respectively. Whenever using single sgRNA or multiplexed sgRNAs, the mRNA expression of HPV18 E7 oncogene was significantly decreased when adding E7-sgRNAs with dsODN, compared to E7-sgRNAs without donor. And the indels% of the targeted sites on the HPV18 E7 gene was markedly increased by adding dsODN with E7-sgRNAs. Finally, we performed GUIDE-Seq to verify that the integrated dsODN could serve as the marker to detect off-target effects in using single or multiplexed two sgRNAs. And we detected fewer on-target reads and off-target sites in multiplexes compared to the single sgRNAs when targeting the GFP and the HPV18 E7 genes. Together, CRISPR-Cas9 system co-transfected with 34nt dsODN concurrently improved the editing efficiency and monitored off-target effects, which might provide new insights in the treatment of HPV infections and related cervical cancer.