Transduction_of_NIH3T3_with_BE3_and_with_sgRNA_vectors_WGS_

Recent reports document that cytidine base editors (CBEs) can induce substantial, genome wide, guide-dependent and independent off-target base modifications (Xin et al., Signal Transduction and Target Therapy, 2019). This complicates making definitive conclusions about the importance of the introduced mutations when using base editing technologies. Therefore, it is critically important to assess the frequency of single nucleotide variations (SNVs) introduced genome wide by the guides that are deployed. We have recently shown that in situ base editing in the mammary gland of mice can be achieved by intraductal injection of high-titer sgRNA-encoding lentiviral vectors in mice with mammary gland-specific expression of the BE3 CBE, which results in targeted gene modification and tumor initiation. In order to profile the off-target spectrum of the sgRNA that were used for this study, we transduced a mouse cell line (NIH3T3) with BE3 and with sgRNA vectors targeting different oncogenes (Akt1 and Pik3ca) and tumor suppressor genes (Trp53 and Pten). Whole genome sequencing with 30x coverage will be performed from their DNA and control DNA from untransduced cells or cells expressing the CBE only. A bioinformatic pipeline for variant calling on the WGS data (Iyer et al., Plos Genetics, 2018) will be then used to demonstrate on-target editing as well as the spectrum of SNVs for all the tested sgRNAs.