Supplementary Figure 6: Targeting of multiple chromosomal loxBTR sequences by Brec1.

(a) HeLa-smurf cells, containing at the PACS1 locus on chromosome 11 a full-length HIV-1 proviral DNA that expresses blue fluorescent protein (BFP), have been previously described²⁹ At a distance of ~ 1 Mb, an additional self-contained reporter construct was inserted at the KDMA2 locus via CRISPR/Cas9 technology. This reporter simultaneously expresses from an HIV-1 LTR promoter the fluorescent protein mKO2 and the HIV trans-activator Tat by use of an ERAV 2A-like sequence. (b) Brec1-mediated provirus recombination results in an excision product that can be detected by PCR (depicted in Fig. 4c). At day 3 post transduction with LV-Brec1, this circular excision product was observed in chromosomal DNA isolated from HeLa-smurf reporter cells. Brec1, LV-Brec1 transduced cells; NC, negative control (non-transduced cells); PC, positive PCR control; H₂O, negative PCR control (omitting DNA template). (c) Analysis of potential aberrant Brec1-mediated loxBTR recombination. To monitor LTR/genomic DNA junctions, and LTR/proviral genome junctions, non-restrictive linear amplification-mediated PCR (nrLAM-PCR) was performed as described previously²⁹, using an mKO2-specific anchor primer (indicated in panel a). In addition to extension products from the reporter construct integrated at the KDM2A locus (product I in panel a), aberrant recombination between distant loxBTR sequences would be expected to yield nrLAM extension products which contain either proviral or 5’-proximal host sequences from the PACS1 locus (products II and III in panel a, respectively). As shown in panel c, PCR analysis with primer pairs designed to amplify upstream integration site sequences from extension products I and III (indicated in panel a) revealed the expected amplification product (asterisk) for primer pair P1/P2, but not for primer pair P3/4, indicating that aberrant recombination (i.e. recombination of the proviral 5’LTR and the LTR of the reporter construct) did not occur. M, DNA marker; PC positive PCR control; mock, nrLAM-PCR template from non-transduced cells; Brec1, nrLAM-PCR template from LV-Brec1 transduced cells; H₂O, negative PCR control (omitting DNA template). Additionally, next generation bulk sequencing of nrLAM amplicons (2x250 bp paired end, Illumina MiSeq platform) yielded 1698 reads with 5’-fragments that spanned the host/loxBTR junction of the reporter construct but none that would indicate aberrant recombination, again indicating that Brec1 expression resulted exclusively in provirus excision.