Dataset Information

Inducing CCR5Δ32/Δ32 Homozygotes in the Human Jurkat CD4+ Cell Line and Primary CD4+ Cells by CRISPR-Cas9 Genome-Editing Technology.

ABSTRACT: C-C chemokine receptor type 5 (CCR5) is the main co-receptor for HIV entry into the target CD4+ cells, and homozygous CCR5Δ32/Δ32 cells are resistant to CCR5-tropic HIV infection. However, the CCR5Δ32/Δ32 homozygous donors in populations are rare. Here we developed a simple approach to induce CCR5Δ32/Δ32 homozygotes through CRISPR-Cas9 genome-editing technology. Designing a pair of single-guide RNA targeting the flank region of the CCR5Δ32 mutation locus, we applied the CRISPR-Cas9 and lentiviral packaging system to successfully convert wild-type CCR5 into CCR5Δ32/Δ32 homozygotes in the human Jurkat CD4+ cell line and primary CD4+ cells, exactly the same as the naturally occurring CCR5Δ32/Δ32 mutation. The successful rate is up to 20% in Jurkat cells but less in primary CD4+ cells. The modified CCR5Δ32/Δ32 CD4+ cells are resistant to CCR5-tropic HIV infection. Whole-genome sequencing revealed no apparent off-target sites. This approach has the promise to promote HIV/AIDS therapy from the only cured unique Berlin patient to a routine autologous cell-based therapy.

SUBMITTER: Qi C

PROVIDER: S-EPMC6005807 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Inducing CCR5Δ32/Δ32 Homozygotes in the Human Jurkat CD4+ Cell Line and Primary CD4+ Cells by CRISPR-Cas9 Genome-Editing Technology.

Qi Chunxia C Li Dan D Jiang Xiangxiang X Jia Xiaopeng X Lu Lingling L Wang Yanfeng Y Sun Jinhuan J Shao Yiming Y Wei Min M

Molecular therapy. Nucleic acids 20180617

C-C chemokine receptor type 5 (CCR5) is the main co-receptor for HIV entry into the target CD4+ cells, and homozygous CCR5Δ32/Δ32 cells are resistant to CCR5-tropic HIV infection. However, the CCR5Δ32/Δ32 homozygous donors in populations are rare. Here we developed a simple approach to induce CCR5Δ32/Δ32 homozygotes through CRISPR-Cas9 genome-editing technology. Designing a pair of single-guide RNA targeting the flank region of the CCR5Δ32 mutation locus, we applied the CRISPR-Cas9 and lentivira ...[more]

PMID: 30195765

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Project description:A cure for HIV-1 remains unattainable as only one case has been reported, a decade ago1,2. The individual-who is known as the 'Berlin patient'-underwent two allogeneic haematopoietic stem-cell transplantation (HSCT) procedures using a donor with a homozygous mutation in the HIV coreceptor CCR5 (CCR5Δ32/Δ32) to treat his acute myeloid leukaemia. Total body irradiation was given with each HSCT. Notably, it is unclear which treatment or patient parameters contributed to this case of long-term HIV remission. Here we show that HIV-1 remission may be possible with a less aggressive and toxic approach. An adult infected with HIV-1 underwent allogeneic HSCT for Hodgkin's lymphoma using cells from a CCR5Δ32/Δ32 donor. He experienced mild gut graft-versus-host disease. Antiretroviral therapy was interrupted 16 months after transplantation. HIV-1 remission has been maintained over a further 18 months. Plasma HIV-1 RNA has been undetectable at less than one copy per millilitre along with undetectable HIV-1 DNA in peripheral CD4 T lymphocytes. Quantitative viral outgrowth assays from peripheral CD4 T lymphocytes show no reactivatable virus using a total of 24 million resting CD4 T cells. CCR5-tropic, but not CXCR4-tropic, viruses were identified in HIV-1 DNA from CD4 T cells of the patient before the transplant. CD4 T cells isolated from peripheral blood after transplantation did not express CCR5 and were susceptible only to CXCR4-tropic virus ex vivo. HIV-1 Gag-specific CD4 and CD8 T cell responses were lost after transplantation, whereas cytomegalovirus-specific responses were detectable. Similarly, HIV-1-specific antibodies and avidities fell to levels comparable to those in the Berlin patient following transplantation. Although at 18 months after the interruption of treatment it is premature to conclude that this patient has been cured, these data suggest that a single allogeneic HSCT with homozygous CCR5Δ32 donor cells may be sufficient to achieve HIV-1 remission with reduced intensity conditioning and no irradiation, and the findings provide further support for the development of HIV-1 remission strategies based on preventing CCR5 expression.

Project description:The discovery and exploitation of the prokaryotic adaptive immunity system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins have revolutionized genetic engineering. CRISPR-Cas tools have enabled extensive genome editing as well as efficient modulation of the transcriptional program in a multitude of organisms. Progress in the development of genetic engineering tools for the genus Clostridium has lagged behind that of many other prokaryotes, presenting the CRISPR-Cas technology an opportunity to resolve a long-existing issue. Here, we applied the Streptococcus pyogenes type II CRISPR-Cas9 (SpCRISPR-Cas9) system for genome editing in Clostridium acetobutylicum DSM792. We further explored the utility of the SpCRISPR-Cas9 machinery for gene-specific transcriptional repression. For proof-of-concept demonstration, a plasmid-encoded fluorescent protein gene was used for transcriptional repression in C. acetobutylicum Subsequently, we targeted the carbon catabolite repression (CCR) system of C. acetobutylicum through transcriptional repression of the hprK gene encoding HPr kinase/phosphorylase, leading to the coutilization of glucose and xylose, which are two abundant carbon sources from lignocellulosic feedstocks. Similar approaches based on SpCRISPR-Cas9 for genome editing and transcriptional repression were also demonstrated in Clostridium pasteurianum ATCC 6013. As such, this work lays a foundation for the derivation of clostridial strains for industrial purposes.ImportanceAfter recognizing the industrial potential of Clostridium for decades, methods for the genetic manipulation of these anaerobic bacteria are still underdeveloped. This study reports the implementation of CRISPR-Cas technology for genome editing and transcriptional regulation in Clostridium acetobutylicum, which is arguably the most common industrial clostridial strain. The developed genetic tools enable simpler, more reliable, and more extensive derivation of C. acetobutylicum mutant strains for industrial purposes. Similar approaches were also demonstrated in Clostridium pasteurianum, another clostridial strain that is capable of utilizing glycerol as the carbon source for butanol fermentation, and therefore can be arguably applied in other clostridial strains.

Dataset Information

Inducing CCR5Δ32/Δ32 Homozygotes in the Human Jurkat CD4+ Cell Line and Primary CD4+ Cells by CRISPR-Cas9 Genome-Editing Technology.

Publications

Inducing CCR5Δ32/Δ32 Homozygotes in the Human Jurkat CD4+ Cell Line and Primary CD4+ Cells by CRISPR-Cas9 Genome-Editing Technology.

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