CRISPR-Cas attack of HIV-1 proviral DNA can cause unintended deletion of surrounding cellular DNA

Several studies indicate that CRISPR-Cas gene-editing systems can be used to inactivate the HIV-1 proviral DNA in infected cells. Such gene editing introduces mutations, mostly small insertions and deletions (indels), at the targeted sites in the HIV genome or causes excision or inversion of the proviral DNA fragment between two target sites. To investigate whether CRISPR-Cas treatment of latently infected T cell lines can also cause large unintended deletions, we designed a PCR-based sequencing strategy with primer binding sites at various positions in the chromosomal DNA surrounding the integrated proviral DNA genome. We, here, demonstrate that both continuous and transient CRISPR-Cas attack on the integrated HIV DNA does not only result in the expected small indels, but also frequently causes much larger deletions that can include flankingcellular DNA sequences. Analysis of the breakpoint junction sites indicates that the deletions were triggered by an initial on-target attack by CRISPR-Cas. Upon continuous CRISPR-Cas treatment, small microhomologies were frequently observed at the junction sites, which indicates that microhomology-mediated end-joining DNA repair is involved in the generation of the large deletions. As the loss of chromosomal sequences may cause oncogenic cell transformation, unintended large deletions form a potential safety risk in clinical application of this antiviral application.