Isolation of gene-edited cells via knock-in of short glycophosphatidylinositol-anchored epitope tags

Zotova, Anastasia; Пичугин, А.В.; Atemasova, Anastasia; Knyazhanskaya, Ekaterina; Lopatukhina, Elena V.; Mitkin, Nikita A.; Holmuhamedov, Ekhson; Gottikh, Marina; Kuprash, Dmitry V.; Filatov, Alexander; Mazurov, Dmitriy

doi:10.1038/s41598-019-40219-z

Cited by 17 publications

(35 citation statements)

References 42 publications

(37 reference statements)

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“…In addition to overexpression, we evaluated HIV-1 infectivity levels in cells with Ku70 depletion. For this purpose, using CRISPR/Cas9 technique and SORTS (Surface Oligopeptide knock-in for Rapid Target Selection) method for isolation of gene-edited cells [42] we generated 293T-Ku70 +/− cells with monoallelic gene knockout (mKO), in which endogenous level of Ku70 was reduced in comparison to that in parental cells—both on protein and RNA level (Fig. 3b, Additional file 1: Figure S3A).…”

Section: Resultsmentioning

confidence: 99%

“…The ~ 100 nt homology arms in a close proximity to DNA cut sites were included into synthetic oligonucleotides together with ~ 18 nt sequences of complementarity to the plasmid templates (Additional file 1: Table S4). Donor DNAs containing short ORF comprised of HA-tag embedded into CD52 and transcriptional terminator from the human β-globin were PCR-amplified from either pJet-CMV-CD5HA2-bglpA plasmid (for K70, Ku80, DNA-PK) or pUCHR-mClover-smAID-P2A-CD5HA2-bglpA plasmid (for LEDGF/p75) [42]. PCR products were gel purified and used to co-transfect cells with CRISPR/Cas9 components.…”

Section: Methodsmentioning

confidence: 99%

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NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase

et al. 2019

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BackgroundHIV-1 integration results in genomic DNA gaps that are repaired by cellular DNA repair pathways. This step of the lentiviral life cycle remains poorly understood despite its crucial importance for successful replication. We and others reported that Ku70 protein of the non-homologous end joining pathway (NHEJ) directly binds HIV-1 integrase (IN). Here, we studied the importance of this interaction for post-integrational gap repair and the recruitment of NHEJ factors in this process.ResultsWe engineered HIV-based pseudovirus with mutant IN defective in Ku70 binding and generated heterozygous Ku70, Ku80 and DNA-PKcs human knockout (KO) cells using CRISPR/Cas9. KO of either of these proteins or inhibition of DNA-PKcs catalytic activity substantially decreased the infectivity of HIV-1 with native IN but not with the mutant one. We used a recently developed qPCR assay for the measurement of gap repair efficiency to show that HIV-1 with mutant IN was defective in DNA post-integrational repair, whereas the wild type virus displayed such a defect only when NHEJ system was disrupted in any way. This effect was present in CRISPR/Cas9 modified 293T cells, in Jurkat and CEM lymphoid lines and in primary human PBMCs.ConclusionsOur data provide evidence that IN recruits DNA-PK to the site of HIV-1 post-integrational repair due to Ku70 binding—a novel finding that explains the involvement of DNA-PK despite the absence of free double stranded DNA breaks. In addition, our data clearly indicate the importance of interactions between HIV-1 IN and Ku70 in HIV-1 replication at the post-integrational repair step.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase

et al. 2019

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“…Next, we generated Jurkat cells with BST2 knockout (KO) using CRISPR/Cas9 technique. To minimize off-target effects and avoid potential biases in retroviral replication related to clonal isolation of gene-edited cells that we reported earlier [36,37], we combined double nicking (DN) approach [38] with a polyclonal isolation of BST2-negative cells after staining with respective antibody and FACS-sorting. As a result, the sorted population of Jurkat cells carried out BST2 null phenotype and did not upregulate BST2 in response to IFNα treatment (Figure 5A, right hand histogram, and B).…”

Section: Resultsmentioning

confidence: 99%

Distinct Requirements for HIV-1 Accessory Proteins during Cell Coculture and Cell-Free Infection

Zotova

Atemasova

Alexey³

et al. 2019

Viruses

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The role of accessory proteins during cell-to-cell transmission of HIV-1 has not been explicitly defined. In part, this is related to difficulties in measuring virus replication in cell cocultures with high accuracy, as cells coexist at different stages of infection and separation of effector cells from target cells is complicated. In this study, we used replication-dependent reporter vectors to determine requirements for Vif, Vpu, Vpr, or Nef during one cycle of HIV-1 cell coculture and cell-free infection in lymphoid and nonlymphoid cells. Comparative analysis of HIV-1 replication in two cell systems showed that, irrespective of transmission way, accessory proteins were generally less required for virus replication in 293T/CD4/X4 cells than in Jurkat-to-Raji/CD4 cell cocultures. This is consistent with a well-established fact that lymphoid cells express a broad spectrum of restriction factors, while nonlymphoid cells are rather limited in this regard. Remarkably, Vpu deletion reduced the level of cell-free infection, but enhanced the level of cell coculture infection and increased the fraction of multiply infected cells. Nef deficiency did not influence or moderately reduced HIV-1 infection in nonlymphoid and lymphoid cell cocultures, respectively, but strongly affected cell-free infection. Knockout of BST2—a Vpu antagonizing restriction factor—in Jurkat producer cells abolished the enhanced replication of HIV-1 ΔVpu in cell coculture and prevented the formation of viral clusters on cell surface. Thus, BST2-tethered viral particles mediated cell coculture infection more efficiently and at a higher level of multiplicity than diffusely distributed virions. In conclusion, our results demonstrate that the mode of transmission may determine the degree of accessory protein requirements during HIV-1 infection.

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“…For SORTS principle and experimental protocol, please, refer to our previous paper (31). Briefly, to initiate knock-in (KI) of gp41 peptide into proviral DNA or cellular gene, 1-2x10 6 CEM, CEM-HIV-1-GFPt cells, activated human PBMC or CD4 + lymphocytes were cotransfected with 3 μg of pcDNA3.3-hCas9, 1 μg of pKS-gRNA plasmid DNAs, and 2 pM of respective PCR or plasmid donor/s.…”

Section: Knock-ins and Sortsmentioning

confidence: 99%

“…We have previously described a novel method for gene-edited cell selection called SORTS (surface oligonucleotide knock-in for rapid target selection), which is based on the targeted biallelic knock-in of epitope tags embedded in CD52 (31). CD52 is the shortest human GPI-anchored protein that can effectively deliver peptides to the plasma membrane, where they serve as makers for fluorescence-activated cell sorting (FACS) isolation of edited cells.…”

Section: Introductionmentioning

confidence: 99%

Engineering T cell resistance to HIV-1 infection via knock-in of peptides from the heptad repeat 2 domain of gp41

Maslennikova

Kruglova

Калиниченко

et al. 2021

Preprint

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Previous studies suggest that short peptides from the heptad repeat 2 (HR2) domain of gp41 expressed on the cell surface are more potent inhibitors of HIV-1 entry than soluble analogs. However, their therapeutic potential has only been examined using lentiviral vectors. Here, we aimed to develop CRISPR/Cas9-based fusion inhibitory peptide knock-in (KI) technology for the generation and selection of HIV-1-resistant T cells. First, we cloned a series of HIV-1 fusion inhibitory peptides and embedded them in CD52, the shortest GPI-anchored protein, which efficiently delivers epitope tags to the cell surface and maintains a sufficient level of KI. Among the seven peptides tested, MT-C34, HP-23L, and 2P23 exhibited significant activity against both cell-free and cell-to-cell HIV-1 infection. Unlike membrane-bound peptides, the shed variant of MT-C34 provided insufficient protection against HIV-1 due to its low concentrations in the culture medium. Using Cas9 plasmids or ribonucleoprotein electroporation and cell sorting with antibodies raised against gp41 peptides, we generated CEM/R5 cells with biallelic KI of MT-C34 (embedded in CD52 for expression in lipid rafts) and 2P23 (N-terminally fused to CXCR4). In combination, these peptides provided a higher level of protection than individual KI. By extending homology arms and substituting PCR donor DNA with a plasmid containing signals for nuclear localization, we achieved KI of MT-C34 into CXCR4 loci and HIV-1 proviral DNA at levels of up to 35% in CEM/R5 cells and increased KI occurrence from undetectable to 4% in CD4 lymphocytes. Regardless of HDR efficiency, sorted cells were completely protected from X4 and R5 strains of HIV-1 and capable of expanding during acute viral replication. Thus, the developed CRISPR/Cas9 platform opens up new possibilities for HIV therapy based on protective peptide KI that, in primary human cells, had previously been considered inefficient.AUTHOR SUMMARYHIV is a human lentivirus that infects CD4-positive immune cells and, when left untreated, manifests in the fatal disease known as acquired immunodeficiency syndrome. Even after suppression with antivirals, HIV persists in the organism as a latent provirus. One way to decrease the viral reservoir is to engineer CD4 lymphocytes that are genetically resistant to HIV-1 infection via entry molecule knockout or expression of different antiviral genes. Peptides from the heptad repeat (HR) domain of gp41 are potent inhibitors of HIV-1 fusion, especially when designed to express on the cell surface. Individual gp41 peptides encoded by therapeutic lentiviral vectors have been evaluated and some have entered clinical trials. However, a CRISPR/Cas9-based gp41 peptide delivery platform that operates through concomitant target gene modification has not yet been developed due to low knock-in (KI) rates in primary cells. Here, we systematically evaluated the antiviral activity of different HR2-peptides cloned into the shortest carrier molecule, CD52. The resulting small-size transgene constructs encoding selected peptides, in combination with improvements to enhance donor vector nuclear import, helped to overcome precise editing restrictions in CD4 lymphocytes. Using KI into CXCR4, we demonstrated different options for target gene modification, effectively protecting edited cells against HIV-1.

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Isolation of gene-edited cells via knock-in of short glycophosphatidylinositol-anchored epitope tags

Cited by 17 publications

References 42 publications

NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase

NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase

Distinct Requirements for HIV-1 Accessory Proteins during Cell Coculture and Cell-Free Infection

Engineering T cell resistance to HIV-1 infection via knock-in of peptides from the heptad repeat 2 domain of gp41

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