Transcriptional activation of the MICA gene with an engineered CRISPR-Cas9 system

Sekiba, Kazuma; Yamagami, Mari; Otsuka, Motoyuki; Suzuki, Tatsunori; Kishikawa, Takahiro; Ishibashi, Rei; Ohno, Motoko; Sato, Masaya; Koike, Kazuhiko

doi:10.1016/j.bbrc.2017.03.076

Cited by 14 publications

(15 citation statements)

References 21 publications

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“…This largely depends on the DNA-binding specificity and affinity of designed zinc-finger and TALE (Transcription activator like effectors) proteins. Recently CRISPR/Cas system has also been used for genome editing experiments [ 1 – 4 ]. Such a knockout technology offers advantage over other approaches such that a specific area of the DNA could be modified with a single dose of administration of these molecules, thereby making permanent change in the genome at once and making it non-functional.…”

Section: Introductionmentioning

confidence: 99%

Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design

et al. 2018

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BackgroundGene knockout technologies involving programmable nucleases have been used to create knockouts in several applications. Gene editing using Zinc-finger nucleases (ZFNs), Transcription activator like effectors (TALEs) and CRISPR/Cas systems has been used to create changes in the genome in order to make it non-functional. In the present study, we have looked into the possibility of using six fingered CompoZr ZFN pair to target the E6 gene of HPV 16 genome.MethodsHPV 16+ve cell lines; SiHa and CaSki were used for experiments. CompoZr ZFNs targeting E6 gene were designed and constructed by Sigma-Aldrich. TALENs targeting E6 and E7 genes were made using TALEN assembly kit. Gene editing was monitored by T7E1 mismatch nuclease and Nuclease resistance assays. Levels of E6 and E7 were further analyzed by RT-PCR, western blot as well as immunoflourescence analyses. To check if there is any interference due to methylation, cell lines were treated with sodium butyrate, and Nocodazole.ResultsAlthough ZFN editing activity in yeast based MEL-I assay was high, it yielded very low activity in tumor cell lines; only 6% editing in CaSki and negligible activity in SiHa cell lines. Though editing efficiency was better in CaSki, no significant reduction in E6 protein levels was observed in immunocytochemical analysis. Further, in silico analysis of DNA binding prediction revealed that some of the ZFN modules bound to sequence that did not match the target sequence. Hence, alternate ZFN pairs for E6 and E7 were not synthesized since no further active sites could be identified by in silico analyses. Then we designed TALENs to target E6 and E7 gene. TALENs designed to target E7 gene led to reduction of E7 levels in CaSki and SiHa cervical cancer cell lines. However, TALEN designed to target E6 gene did not yield any editing activity.ConclusionsOur study highlights that designed nucleases intended to obtain bulk effect should have a reasonable editing activity which reflects phenotypically as well. Nucleases with low editing efficiency, intended for generation of knockout cell lines nucleases could be obtained by single cell cloning. This could serve as a criterion for designing ZFNs and TALENs.Electronic supplementary materialThe online version of this article (10.1186/s12935-018-0666-0) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design

et al. 2018

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“…Flow cytometry was performed as described previously 21 . Briefly, cells were hybridized with anti-MICA (1:500; R&D Systems, Minneapolis, MN) or an isotype control IgG (1:500; R&D Systems) for 40 min at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

“…While we have reported several methods of increasing MICA protein levels using microRNAs, antisense nucleotides, and the clustered regularly interspaced short palindromic repeats-Cas9 system 19 – 21 , methods of regulating MICA/B shedding are needed to enhance immunity and develop oncogenesis-prevention strategies. Because drug repositioning enables translation of laboratory discoveries to the clinic 22 , we screened a library of protease inhibitors, most of which are under development for clinical use, to identify inhibitors of MICA/B shedding.…”

Section: Introductionmentioning

confidence: 99%

The fatty-acid amide hydrolase inhibitor URB597 inhibits MICA/B shedding

Sekiba

Otsuka

Seimiya

et al. 2020

Sci Rep

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MICA/B proteins are expressed on the surface of various types of stressed cells, including cancer cells. Cytotoxic lymphocytes expressing natural killer group 2D (NKG2D) receptor recognize MICA/B and eliminate the cells. However, cancer cells evade such immune recognition by inducing proteolytic shedding of MICA/B proteins. Therefore, preventing the shedding of MICA/B proteins could enhance antitumor immunity. Here, by screening a protease inhibitor library, we found that the fatty-acid amide hydrolase (FAAH) inhibitor, URB597, suppresses the shedding of MICA/B. URB597 significantly reduced the soluble MICA level in culture medium and increased the MICA level on the surface of cancer cells. The effect was indirect, being mediated by increased expression of tissue inhibitor of metalloproteinases 3 (TIMP3). Knockdown of TIMP3 expression reversed the effect of URB597, confirming that TIMP3 is required for the MICA shedding inhibition by URB597. In contrast, FAAH overexpression reduced TIMP3 expression and the cell-surface MICA level and increased the soluble MICA level. These results suggest that inhibition of FAAH could prevent human cancer cell evasion of immune-mediated clearance.

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“…Although not applied in pre-clinical studies, a number of early works also suggest that dCas9-based methods can help circumvent other major tumor immunoevasion mechanisms. For instance, impairment of Treg immunosuppressive function by dCas9-KRAB-mediated forkhead box P3 (FoxP3) transcriptional repression, 139 , 140 dCas9-SAM-mediated enhancement of neoplastic transformation markers, such as MICA, 141 , 142 and dCas9-KRAB-directed repression of immunosuppressive cytokine receptors 143 have demonstrated that dCas9-mediated epigenetic engineering has potential in multiple facets of cancer immunotherapy. Ultimately, dCas9-mediated epigenetic editing to improve tumor immunogenicity and/or boost the host’s anti-tumor immune response opens new therapeutic avenues, particularly in combination with ICI or ACT therapies ( Figure 2 ).…”

Section: Epigenetic Editing By Crispr-dcas9 Can Improve Anti-tumor Host Immunitymentioning

confidence: 99%

Reprogramming the anti-tumor immune response via CRISPR genetic and epigenetic editing

Alves

Taifour

Dolcetti

et al. 2021

Molecular Therapy - Methods & Clinical Development

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Precise clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genetic and epigenetic manipulation of the immune response has become a promising immunotherapeutic approach toward combating tumorigenesis and tumor progression. CRISPR-based immunologic reprograming in cancer therapy comprises the locus-specific enhancement of host immunity, the improvement of tumor immunogenicity, and the suppression of tumor immunoevasion. To date, the ex vivo re-engineering of immune cells directed to inhibit the expression of immune checkpoints or to express synthetic immune receptors (chimeric antigen receptor therapy) has shown success in some settings, such as in the treatment of melanoma, lymphoma, liver, and lung cancer. However, advancements in nuclease-deactivated CRISPR-associated nuclease-9 (dCas9)-mediated transcriptional activation or repression and Cas13-directed gene suppression present novel avenues for the development of tumor immunotherapies. In this review, the basis for development, mechanism of action, and outcomes from recently published Cas9-based clinical trial (genetic editing) and dCas9/Cas13-based pre-clinical (epigenetic editing) data are discussed. Lastly, we review cancer immunotherapy-specific considerations and barriers surrounding use of these approaches in the clinic.

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Transcriptional activation of the MICA gene with an engineered CRISPR-Cas9 system

Cited by 14 publications

References 21 publications

Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design

Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design

The fatty-acid amide hydrolase inhibitor URB597 inhibits MICA/B shedding

Reprogramming the anti-tumor immune response via CRISPR genetic and epigenetic editing

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