Characterization of genomic rearrangements in RPE-1 cells with and without DSB in the ABCB1 promoter

The discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) and its development as a genome editing tool has revolutionized the field of molecular biology. In the DNA damage field, CRISPR has brought an alternative to induce endogenous double-strand breaks (DSB) at desired genomic locations and study the DNA damage response and its consequences. Many systems for sgRNA delivery have been reported in order to efficiency generate this DSB, including lentiviral vectors. However, some of the consequences of these systems are yet not well understood. Here we report that lentiviral-based sgRNA vectors can integrate into the endogenous genomic target location, leading to undesired activation of the target gene. By generating a DSB in the regulatory region of the ABCB1 gene using a lentiviral sgRNA vector, we can induce the formation of taxol-resistant colonies. We show that these colonies upregulated ABCB1 via integration of the EEF1A1 and the U6 promoters from the sgRNA vector. We believe that this is an unreported CRISPR/Cas9 artefact that researchers need to be aware of when using lentiviral vectors for genome editing. We have performed Targeted Locus Amplification (TLA), a chromosome conformation capture-based technique to selectively aplify and sequence DNA flanking the ABCB1 promoter. We employed two cell lines: RPE-1 parental and an RPE-1 clone (sg6C9) with a Cas9-DSB induced in ABCB1 (Sample 1 and 2). We have also performed ChIP seq of RPE-1 parental and seven clones with a DSB-induced in ABCB1 which are taxol resistant: sg6C1, sg6C9, sg11C3, sg11C4, sg11C6, sg12C5, sg11C4 (Samples 3 to 11).