Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity


Current combination antiretroviral therapies (cART) efficiently suppress HIV-1 reproduction in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. To generate an antiviral agent capable of eradicating the provirus from infected cells, we employed 145 cycles of substrate-linked directed evolution to evolve a recombinase (Brec1) that site-specifically recognizes a 34-bp sequence present in the long terminal repeats (LTRs) of the majority of the clinically relevant HIV-1 strains and subtypes. Brec1 efficiently, precisely and safely removes the integrated provirus from infected cells and is efficacious on clinical HIV-1 isolates in vitro and in vivo, including in mice humanized with patient-derived cells. Our data suggest that Brec1 has potential for clinical application as a curative HIV-1 therapy.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Figure 1: Highly conserved recombinase target site loxBTR in HIV-1 LTRs.
Figure 2: Brec1 evolution.
Figure 3: Brec1 recombination is efficient and specific in bacteria.
Figure 4: Brec1 recombination in mammalian cells.
Figure 5: Brec1 deletes the HIV-1 provirus from patient-derived primary T cells.
Figure 6: Brec1-mediated antiviral effects in NSG mice engrafted with human CD34+ HSC.

Accession codes

Primary accessions


Referenced accessions


Protein Data Bank


  1. Passaes, C.P. & Sáez-Cirión, A. HIV cure research: advances and prospects. Virology 454–455, 340–352 (2014).

    Article  PubMed  CAS  Google Scholar 

  2. Maartens, G., Celum, C. & Lewin, S.R. HIV infection: epidemiology, pathogenesis, treatment, and prevention. Lancet 384, 258–271 (2014).

    Article  PubMed  Google Scholar 

  3. Fauci, A.S., Marston, H.D. & Folkers, G.K. An HIV cure: feasibility, discovery, and implementation. J. Am. Med. Assoc. 312, 335–336 (2014).

    Article  CAS  Google Scholar 

  4. Mbonye, U. & Karn, J. Transcriptional control of HIV latency: cellular signaling pathways, epigenetics, happenstance and the hope for a cure. Virology 454–455, 328–339 (2014).

    Article  PubMed  CAS  Google Scholar 

  5. Battistini, A. & Sgarbanti, M. HIV-1 latency: an update of molecular mechanisms and therapeutic strategies. Viruses 6, 1715–1758 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Chun, T.W., Davey, R.T. Jr., Engel, D., Lane, H.C. & Fauci, A.S. Re-emergence of HIV after stopping therapy. Nature 401, 874–875 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Akay, C. et al. Antiretroviral drugs induce oxidative stress and neuronal damage in the central nervous system. J. Neurovirol. 20, 39–53 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Richman, D.D. Antiviral drug resistance. Antiviral Res. 71, 117–121 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Pinoges, L. et al. Risk factors and mortality associated with resistance to first-line antiretroviral therapy: multicentric cross-sectional and longitudinal analyses. J. Acquir. Immune Defic. Syndr. 68, 527–535 (2015).

    Article  CAS  PubMed  Google Scholar 

  10. Petoumenos, K. et al. D:A:D Study Group. Predicting the short-term risk of diabetes in HIV-positive patients: the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study. J. Int. AIDS Soc. 15, 17426 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Nachega, J.B. et al. HIV treatment adherence, drug resistance, virologic failure: evolving concepts. Infect. Disord. Drug Targets 11, 167–174 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Rappold, M. et al. Treatment modification in HIV-Infected individuals starting antiretroviral therapy between 2011 and 2014. J. Int. AIDS Soc. 17 (suppl. 3), 19768 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Schackman, B.R. et al. The lifetime cost of current human immunodeficiency virus care in the United States. Med. Care 44, 990–997 (2006).

    Article  PubMed  Google Scholar 

  14. Beltrán, L.M. et al. Influence of immune activation and inflammatory response on cardiovascular risk associated with the human immunodeficiency virus. Vasc. Health Risk Manag. 11, 35–48 (2015).

    PubMed  PubMed Central  Google Scholar 

  15. Sarkar, I., Hauber, I., Hauber, J. & Buchholz, F. HIV-1 proviral DNA excision using an evolved recombinase. Science 316, 1912–1915 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Qu, X. et al. Zinc-finger-nucleases mediate specific and efficient excision of HIV-1 proviral DNA from infected and latently infected human T cells. Nucleic Acids Res. 41, 7771–7782 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ebina, H., Misawa, N., Kanemura, Y. & Koyanagi, Y. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci. Rep. 3, 2510 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Hu, W. et al. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. Proc. Natl. Acad. Sci. USA 111, 11461–11466 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Surendranath, V., Chusainow, J., Hauber, J., Buchholz, F. & Habermann, B.H. SeLOX--a locus of recombination site search tool for the detection and directed evolution of site-specific recombination systems. Nucleic Acids Res. 38, W293–W298 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Buchholz, F. & Stewart, A.F. Alteration of Cre recombinase site specificity by substrate-linked protein evolution. Nat. Biotechnol. 19, 1047–1052 (2001).

    Article  CAS  PubMed  Google Scholar 

  21. Santoro, S.W. & Schultz, P.G. Directed evolution of the site specificity of Cre recombinase. Proc. Natl. Acad. Sci. USA 99, 4185–4190 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sauer, B. & McDermott, J. DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages. Nucleic Acids Res. 32, 6086–6095 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Karimova, M. et al. Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system. Nucleic Acids Res. 41, e37 (2013).

    Article  CAS  PubMed  Google Scholar 

  24. Buchholz, F. & Hauber, J. In vitro evolution and analysis of HIV-1 LTR-specific recombinases. Methods 53, 102–109 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Abi-Ghanem, J. et al. Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach. Nucleic Acids Res. 41, 2394–2403 (2013).

    Article  CAS  PubMed  Google Scholar 

  26. Van Duyne, G.D. A structural view of cre-loxP site-specific recombination. Annu. Rev. Biophys. Biomol. Struct. 30, 87–104 (2001).

    Article  CAS  PubMed  Google Scholar 

  27. Ennifar, E., Meyer, J.E., Buchholz, F., Stewart, A.F. & Suck, D. Crystal structure of a wild-type Cre recombinase-loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation. Nucleic Acids Res. 31, 5449–5460 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Suerth, J.D., Maetzig, T., Galla, M., Baum, C. & Schambach, A. Self-inactivating alpharetroviral vectors with a split-packaging design. J. Virol. 84, 6626–6635 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Hauber, I. et al. Highly significant antiviral activity of HIV-1 LTR-specific tre-recombinase in humanized mice. PLoS Pathog. 9, e1003587 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Perez, E.E. et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26, 808–816 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Holt, N. et al. Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat. Biotechnol. 28, 839–847 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Whitney, J.B. et al. Rapid seeding of the viral reservoir prior to SIV viraemia in rhesus monkeys. Nature 512, 74–77 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Taylor, B.S., Sobieszczyk, M.E., McCutchan, F.E. & Hammer, S.M. The challenge of HIV-1 subtype diversity. N. Engl. J. Med. 358, 1590–1602 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Crowell, T.A. et al. Hospitalization rates and reasons among HIV elite controllers and persons with medically controlled HIV infection. J. Infect. Dis. 211, 1692–1702 (2015).

    Article  PubMed  Google Scholar 

  35. Suzuki, E. & Nakayama, M. VCre/VloxP and SCre/SloxP: new site-specific recombination systems for genome engineering. Nucleic Acids Res. 39, e49 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Tebas, P. et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N. Engl. J. Med. 370, 901–910 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Pattanayak, V., Ramirez, C.L., Joung, J.K. & Liu, D.R. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat. Methods 8, 765–770 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Gabriel, R. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat. Biotechnol. 29, 816–823 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. Tsai, S.Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

    Article  CAS  PubMed  Google Scholar 

  40. Wang, X. et al. Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat. Biotechnol. 33, 175–178 (2015).

    Article  CAS  PubMed  Google Scholar 

  41. Kim, H. & Kim, J.S. A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15, 321–334 (2014).

    Article  CAS  PubMed  Google Scholar 

  42. Josefsson, L. et al. Majority of CD4+ T cells from peripheral blood of HIV-1-infected individuals contain only one HIV DNA molecule. Proc. Natl. Acad. Sci. USA 108, 11199–11204 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Josefsson, L. et al. Single cell analysis of lymph node tissue from HIV-1 infected patients reveals that the majority of CD4+ T-cells contain one HIV-1 DNA molecule. PLoS Pathog. 9, e1003432 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Ringrose, L., Chabanis, S., Angrand, P.O., Woodroofe, C. & Stewart, A.F. Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances. EMBO J. 18, 6630–6641 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Santarosa, M. & Ashworth, A. Haploinsufficiency for tumour suppressor genes: when you don't need to go all the way. Biochim. Biophys. Acta 1654, 105–122 (2004).

    CAS  PubMed  Google Scholar 

  46. Archin, N.M. et al. Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy. Nature 487, 482–485 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Caskey, M. et al. Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117. Nature 522, 487–491 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Blankson, J.N., Siliciano, J.D. & Siliciano, R.F. Finding a cure for human immunodeficiency virus-1 infection. Infect. Dis. Clin. North Am. 28, 633–650 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Scherer, L.J. & Rossi, J.J. Ex vivo gene therapy for HIV-1 treatment. Hum. Mol. Genet. 20, R100–R107 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Herrera-Carrillo, E. & Berkhout, B. Bone Marrow Gene Therapy for HIV/AIDS. Viruses 7, 3910–3936 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Deeks, S.G. HIV: Shock and kill. Nature 487, 439–440 (2012).

    Article  CAS  PubMed  Google Scholar 

  52. Katlama, C. et al. Barriers to a cure for HIV: new ways to target and eradicate HIV-1 reservoirs. Lancet 381, 2109–2117 (2013).

    Article  CAS  PubMed  Google Scholar 

  53. Münch, R.C. et al. Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors. Nat. Commun. 6, 6246 (2015).

    Article  PubMed  CAS  Google Scholar 

  54. Chomont, N. et al. HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nat. Med. 15, 893–900 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Anastassiadis, K. et al. Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice. Dis. Model. Mech. 2, 508–515 (2009).

    Article  CAS  PubMed  Google Scholar 

  56. Langmead, B. & Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Li, H. et al. 1000 Genome Project Data Processing Subgroup. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  58. Prüfer, K. et al. PatMaN: rapid alignment of short sequences to large databases. Bioinformatics 24, 1530–1531 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  59. Waterhouse, A.M., Procter, J.B., Martin, D.M.A., Clamp, M. & Barton, G.J. Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


The authors are indebted to M. Karimova (TU Dresden; TUD) for providing reagents, R. Kuhnert (TUD) for help with mouse work, A. Käßner-Frensel (TUD) for SKY analyses, R. Naumann (Max Planck Institute for Molecular Cell Biology and Genetics; MPI-CBG) for the generation of transgenic mice, S. Winkler (MPI-CBG) and A. Dahl (DFG-Forschungszentrum für Regenerative Therapien Dresden) for DNA sequencing, D. Dubrau (Heinrich Pette Institute) for initial ASLV-vector construction, G. Pilnitz-Stolze, B. Weseloh and B. Abel (Heinrich Pette Institute) for technical assistance, and D. Indenbirken and M. Alawi (Heinrich Pette Institute) for next-generation sequencing of nrLAM-PCR products. This work was supported by the DFG (BU 1400/7-1), the BMBF (GO-Bio FKZ 0315090), the Else Kröner-Fresenius-Stiftung (2010_A82), and the “Viral Latency” program at the Heinrich Pette Institute–Leibniz Institute for Experimental Virology, Hamburg. The reporter plasmid pCAGGS-loxP-mCherry-loxP-EGFP, where a floxed mCherry gene was inserted between the CAG enhancer/promoter module and the EGFP gene, was a generous gift from E.M. Tanaka (DFG-Forschungszentrum für Regenerative Therapien Dresden). The mammalian expression vector pCAGGS-IRES-puro was a generous gift from K. Anastassiadis (TUD).

Author information

Authors and Affiliations



J.K., I.H. and J.C. designed experiments, performed experiments evaluated data and wrote the manuscript. C.S., D.C., N.B., H.H.-S., U.C.L. and K.H. designed experiments, performed experiments and evaluated data. A.G., M.P.-R. and V.S. designed experiments, performed computational analyses and evaluated data. E.S. designed experiments and evaluated data. J.A.-G. and M.T.P. performed structure-based analyses and evaluated data. A.S., C.L. and J.v.L. provided essential reagents. J.H. and F.B. designed the study, directed the study, evaluated data and wrote the manuscript.

Corresponding authors

Correspondence to Joachim Hauber or Frank Buchholz.

Ethics declarations

Competing interests

J.K., J.C., J.H. and F.B. are inventors on a patent application regarding Brec1 (EP 14183277).

Integrated supplementary information

Supplementary Figure 1 Substrate-linked protein evolution components.

(a) loxBTR subsites used for the combinational directed-evolution process to generate Brec1. Sequence differences (compared to the pool of employed related recombinase target sites) in the enzyme bound half sites of the Brec1 target site loxBTR and loxBTR subsites are marked in red or green for the reverse complementary half site. The spacer sequences are shown in grey. (b) Evolution vector pEVO-recomb-target. In E. coli, the recombinase is expressed from the pBAD promoter upon induction with L-arabinose. The vector also contains the regulatory gene araC and a chloramphenicol resistance marker (Cmr). Recombination at the target sites leads to deletion of the intervening region containing a unique NdeI restriction site; following DNA cleavage with NdeI, successful candidates were retrieved by PCR using primers P1 and P3. Primer P2 was employed for PCR amplification of the full-length recombinase coding sequence after DNA shuffling. BsrGI and XbaI restriction sites flanking the recombinase coding sequence facilitate convenient cloning of the recombinases and simple assaying of recombination efficiency in bacteria. The sizes of the unrecombined and recombined (Δ) forms of the vector are indicated.

Supplementary Figure 2 Amino acid sequence of Brec1.

The NLS sequence, added for nuclear localization of Brec1 in mammalian cells, is shown in blue. Mutations and their corresponding amino acid number in the Cre sequence are shown in red, evolutionary conserved mutations are shown in bold. DNA contact residues based on the Cre crystal structure are underlined.

Supplementary Figure 3 Brec1 efficiency and specificity recombines loxBTR in a genomic context.

(a) Schematic representation of recombination of reporter constructs stably integrated in the genome. After recombination, PCR results in a 0.7 kb product (one triangle), while non-recombined template produces a 1.8 kb PCR product (two triangles). (b) Reporter HeLa cell lines with stably integrated loxBTR target sites were transfected with recombinase expression plasmid. Genomic DNA was isolated and assayed for recombination by PCR using primers F and R that anneal to the integrated DNA regions: -ctr, transfection of wild type cells (lane 1); +ctr, transfection with recombined reporter (lane 2); lanes 3-6 show results from cells carrying genomic loxBTR sites transfected with the indicated expression plasmids; M, DNA marker lane. (c) Reporter HeLa cell lines with stably integrated target sites loxBTR, loxP or loxLTR were transfected with recombinase Brec1 expression plasmid (lanes 1-3). Genomic DNA was isolated and assayed for recombination by PCR using primers F and R. Controls for cells harboring the loxP or LoxLTR reporters, transfected with Cre or Tre recombinase expression vectors are shown in lanes 4 and 5.

Supplementary Figure 4 Safety evaluations.

Exponentially growing Jurkat T cells were transduced with LV-cBrec1, encoding constitutively expressed Brec1 recombinase, with LV-Brec1 (Tat-inducible promotor configuration), or mock transduced, respectively. Transduced GFP+ cells were enriched by FACS. (a) Transduced cells (3 x 106) were seeded into dishes and were grown for 11 days. At indicated time point cells were counted and total cell numbers were calculated. (b) Cell cycle progression monitored by DNA staining at week 6 of Brec1 expression. Jurkat cells were harvested, fixed, and DNA content was stained with propidium iodide. Signals were measured by analytic flow cytometry. (c) Primary human CD4+ T cells were transduced with LV-cBrec1 (constitutive promoter configuration), with LV-Ctr (encoding GFP only), or were mock transduced. Transduced GFP+ cells were enriched by FACS and cultured for two weeks. Subsequently, expression of Brec1 or GAPDH (control) was analyzed by RT-PCR. (d) Analysis of apoptosis in primary human CD4+ T cells at week two post transduction by Annexin V/propidium iodide staining. (e) Functionality of LV-cBrec1 transduced primary CD4+ T cells at two weeks post transduction tested by flow cytometric analysis of CD154 and IFN expression after 3 hours of PMA/ionomycin stimulation. (f) Secretion pattern of Th1-, Th2- and Th17-specific cytokines in primary transduced CD4+ T cells after PMA/Ionomycin stimulation two weeks after transduction as determined by multiplex ELISA. The pattern matches that of non-transduced cells (mock). (g) IL4-specific Elispot analysis of human primary mock or LV-cBrec1 transduced CD4+ T cells after PMA/Ionomycin stimulation two weeks after transduction. (h) CD34+ HSC were mock-transduced or transduced with LV-Ctr, LV-Brec1, or LV-cBrec and 100 cells were seeded into cytokine-containing methyl-cellulose. Culture dishes were incubated for 14 days before counting colonies.

Supplementary Figure 5 Brec1 specificity assays.

(a) Nucleotide sequences of human genomic sites that most closely resemble the loxBTR sequence and their locations in the human genome. Sequences are aligned to loxBTR. Nucleotides that differ from loxBTR are shown in red. (b) Agarose gel showing the activity of Brec1 on loxBTR (lanes 1 and 5, positive control) and the lack of activity for the six loxBTR-like human genomic sites HGS1 – HGS6 (lanes 2-4 and 6-8). E. coli cells harboring the pEVO vector containing the Brec1 coding sequence and indicated target sites were grown at 1 mg/ml L-arabinose. Recombination was assayed by restriction enzyme digest, resulting in a smaller fragment for recombined (one triangle) and a larger fragment for non-recombined substrate (two triangles). M, DNA marker. (c) SKY (spectral karyotyping) analysis of metaphase spreads isolated from primary human CD4+ T cells constitutively expressing Brec1 (LV-cBrec1). An RGB display of the 24-colour SKY hybridization of a representative normal metaphase is shown. Number of analyzed chromosome spreads: 27.

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.

Supplementary Figure 7 Generation and analysis of transgenic Brec1 mice.

(a) Schematic representation of the Brec1 expression cassette. Brec1 is expressed from the cytomegalovirus (CMV) immediate early enhancer-chicken beta-actin hybrid (CAG) promoter. Breeding strategy following generation of Brec1 founder mice by pronuclear injection: WT, wild type mouse; TGM, transgenic mouse. The number of offspring and the TG carriers indicated for the first and second generation show that the transgene is inherited with a Mendelian ratio. (b) Agarose gel showing PCR-based genotyping of one founder (lane 2) and three transgenic offspring (lanes 3-5) using Brec1-specific primers. WT, wild type control (lane 1); M, DNA marker lane. (c) Agarose gel showing PCR-based analysis of cDNA generated by reverse transcription of mRNA isolated from WT animal (control, lane 1) and three transgenic Brec1 mice (TGM1-TGM3, lanes 2-7): +, reverse transcriptase added to RT reaction; -, no-reverse transcriptase control. (d) Schematic representation of the lentiviral reporter construct. Important elements are indicated. The vector backbone has been previously described in detail29 (e) Schematic drawing of the primer binding sites within the lentiviral reporter construct. Important elements are indicated and the size of the expected amplicon for primers P3 and P4 is shown. Brec1-mediated recombination leads to the formation of a circular excision product that can be detected employing primer P1 and P2. (f) Schematic of the 600 bp amplification product for primers P1 and P2. A sequencing read is shown confirming the expected sequence. The loxBTR sequence is marked. (g) Confirmation of lentiviral integration of the reporter construct. PCR assays for indicated animals is provided. The 1.7 kb fragment resulting from amplification with primers P3 and P4 is marked by an asterisk. (h) Confirmation of Brec-1 mediated recombination. Gel showing PCR analysis using DNA isolated from cells of indicated animals. The product showing the recombined band is indicated with P1/P2.

Supplementary Figure 8 Analysis of Brec1 activity in mice engrafted with CD4+ T cells isolated from HIV infected patients.

(a) Monitoring of viral load (red dots) and percentage of human CD45+CD4+ cells (blue dots) in hu-Rag2 mice engrafted with negative control vector (LV-Ctr) transduced primary CD4+ T cells isolated from PBMC of HIV infected patients. (b) Antiviral activity and host cell protection in hu-Rag2 mice engrafted with LV-Brec1 transduced patient-derived CD4+ T lymphocytes. Human cells in PBMC, spleen and liver of the respective Brec1-expressing animal was determined at necropsy by FACS using single cell suspensions (bar chart).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Table 3 (PDF 8079 kb)

Supplementary Table 1

Representation of recombinase target sites loxP, loxLTR, and loxBTR in HIV-1 LTR sequences of the most prevalent subtypes (XLS 27 kb)

Supplementary Table 2

Conserved amino acids emerging during substrate-linked evolution (XLS 30 kb)

Supplementary Table 4 (XLS 222 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Karpinski, J., Hauber, I., Chemnitz, J. et al. Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity. Nat Biotechnol 34, 401–409 (2016).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing