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

Karpinski, Janet; Hauber, Ilona; Chemnitz, Jan; Schäfer, Carola; Paszkowski‐Rogacz, Maciej; Chakraborty, Deboyoti; Beschorner, Niklas; Hofmann-Sieber, Helga; Lange, Ulrike; Grundhoff, Adam; Hackmann, Karl; Schröck, Evelin; Abi-Ghanem, Joséphine; Pisabarro, M. Teresa; Surendranath, Vineeth; Schambach, Axel; Lindner, Christoph; Lunzen, Jan van; Hauber, Joachim; Buchholz, Frank

doi:10.1038/nbt.3467

Cited by 110 publications

(151 citation statements)

References 59 publications

Supporting

Mentioning

110

Contrasting

Order By: Relevance

“…Unlike mutations induced by programmable nucleases such as ZFNs, TALENs or Cas9, recCas9 deletion is not dependent on error-prone forms of DSB repair and should not be prone to undesired chromosomal rearrangements such as translocations. Non-programmable recombinase-mediated deletions have already proven effective at removing latent HIV provirus from infected hematopoetic stem cells (23), or unwanted vector backbone resulting from ex vivo gene therapy (68). Furthermore, the requirement of four separate recCas9 guide RNA-programmed binding events as well as a matching dinucleotide core in the recombination substrates may reduce the likelihood of off-target modifications commonly observed in nuclease-mediated mutagenesis.…”

Section: Discussionmentioning

confidence: 99%

A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells

et al. 2016

View full text Add to dashboard Cite

We describe the development of ‘recCas9’, an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive dCas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. We show that these recombinases can operate on DNA sites in mammalian cells identical to genomic loci naturally found in the human genome in a manner that is dependent on the guide RNA sequences. DNA sequencing reveals that recCas9 catalyzes guide RNA-dependent recombination in human cells with an efficiency as high as 32% on plasmid substrates. Finally, we demonstrate that recCas9 expressed in human cells can catalyze in situ deletion between two genomic sites. Because recCas9 directly catalyzes recombination, it generates virtually no detectable indels or other stochastic DNA modification products. This work represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous cellular machinery or cell state. Current and future generations of recCas9 may facilitate targeted agricultural breeding, or the study and treatment of human genetic diseases.

show abstract

Section: Discussionmentioning

confidence: 99%

A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells

et al. 2016

View full text Add to dashboard Cite

show abstract

“…This multiplex approach can yield increased suppression of HIV-1 infection with a decreased GFP intensity, with gRNAs targeted to the LTR shown to be most effective [137]. Finally, recent reports of a designer Brec recombinase efficiently targeting and excising LTRs on scale holds promise for tackling latent proviral HIV [138]. …”

Section: Applicationsmentioning

confidence: 99%

Genome-Edited T Cell Therapies

Delhove

Qasim

2017

Curr Stem Cell Rep

View full text Add to dashboard Cite

Purpose of ReviewAlternative approaches to conventional drug-based cancer treatments have seen T cell therapies deployed more widely over the last decade. This is largely due to their ability to target and kill specific cell types based on receptor recognition. Introduction of recombinant T cell receptors (TCRs) using viral vectors and HLA-independent T cell therapies using chimeric antigen receptors (CARs) are discussed. This article reviews the tools used for genome editing, with particular emphasis on the applications of site-specific DNA nuclease mediated editing for T cell therapies.Recent FindingsGenetic engineering of T cells using TCRs and CARs with redirected antigen-targeting specificity has resulted in clinical success of several immunotherapies. In conjunction, the application of genome editing technologies has resulted in the generation of HLA-independent universal T cells for allogeneic transplantation, improved T cell sustainability through knockout of the checkpoint inhibitor, programmed cell death protein-1 (PD-1), and has shown efficacy as an antiviral therapy through direct targeting of viral genomic sequences and entry receptors.SummaryThe combined use of engineered antigen-targeting moieties and innovative genome editing technologies have recently shown success in a small number of clinical trials targeting HIV and hematological malignancies and are now being incorporated into existing strategies for other immunotherapies.

show abstract

“…They created a HIV specific Tre recombinase (HIV LTR-specific recombinase) from the Cre (Causes Recombination) recombinase to modify its specificity from loxP site to HIV LTR and recognizes an asymmetric sequence within an HIV LTR. They recently show that the Tre recombinase could excise patient derived HIV provirus from the genome of cell line as well as primary cells [70,71]. Qu et al developed a ZFN to excise integrated HIV provirus.…”

Section: Anti-hiv Genes Provide Resistance To Hiv Infectionmentioning

confidence: 99%

Stem cell-based therapies for HIV/AIDS

Pernet

Yadav

2016

Advanced Drug Delivery Reviews

View full text Add to dashboard Cite

One of the current focuses in HIV/AIDS research is to develop a novel therapeutic strategy that can provide a life-long remission of HIV/AIDS without daily drug treatment and ultimately a cure for HIV/AIDS. Hematopoietic stem cell based anti-HIV gene therapy aims to reconstitute patient immune system by transplantation of genetically engineered hematopoietic stem cells with anti-HIV genes. Hematopoietic stem cells can self renew, proliferate and differentiate into mature immune cells. In theory, anti-HIV gene modified hematopoietic stem cells can continuously provide HIV resistant immune cells throughout the life of a patient. Therefore, hematopoietic stem cell based anti-HIV gene therapy has a great potential to provide a life-long remission of HIV/AIDS by a single treatment. Here, we provide a comprehensive review of the recent progress of developing anti-HIV genes, genetic modification of hematopoietic stem progenitor cells, engraftment and reconstitution of anti-HIV gene modified immune cells, HIV inhibition in vitro and in vivo animal models, and human clinical trials.

show abstract

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

Cited by 110 publications

References 59 publications

A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells

A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells

Genome-Edited T Cell Therapies

Stem cell-based therapies for HIV/AIDS

Contact Info

Product

Resources

About