Site-specific nuclease single-cell assay targeting gene regulatory elements to silence gene expression

a gene regulatory element and single-cell technology, applied in the direction of hydrolases, microorganism testing/measurement, biochemistry apparatus and processes, etc., can solve the problem that current sirna and 3c-based experimental approaches cannot be applied to multigene complexes, cannot reveal the necessity of chromosomal contacts for cotranscription, and cannot eliminate the transcriptional activity of the gene of interest. , to achieve the effect of reducing the capacity of a gene loop, o

Inactive Publication Date: 2019-04-18
CSIR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020]In another embodiment, the transcriptional activity of the gene of interest may be abrogated or prevented by recruitment of proteins involved in a repair process to the site of the double stranded break, which when bound to the chromatin or DNA at the site of the double stranded break obstruct chromosomal contacts. Alternatively, the transcriptional activity of the gene of interest may be abrogated or prevented by means of enhancement of mobility of the region of chromatin or DNA containing the double stranded break, which results in a reduction in the capacity of a gene loop to engage in chromosomal contact. In a further alternative, the transcriptional activity of the gene of interest may be abrogated or prevented through the loss of structural integrity of a gene loop, which results in the abrogation of chromosomal contact.

Problems solved by technology

Therefore, siRNA-approaches fail to reveal cell-to-cell variability.
Off-target effects represent another major challenge in the use of siRNA as gene knockdown tools.
However, these approaches do not reveal the necessity of chromosomal contacts for cotranscription in multigene complexes.
Current siRNA and 3C-based experimental approaches cannot be applied to multigene complexes where all interacting genes are activated by the same transcription factor.
However, both 3C and FISH approaches fail to reveal the necessity of chromosomal contacts for co-transcription of these interacting genes.
Importantly, owing to variegated gene expression (Noordermeer et al., 2011), this can only be achieved with a single cell approach.

Method used

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  • Site-specific nuclease single-cell assay targeting gene regulatory elements to silence gene expression
  • Site-specific nuclease single-cell assay targeting gene regulatory elements to silence gene expression
  • Site-specific nuclease single-cell assay targeting gene regulatory elements to silence gene expression

Examples

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Effect test

example 1

[0132]Cell Culture

[0133]Early passage HUVECs from pooled donors (Lonza) were grown to ˜80% confluence in Endothelial Basal Medium-2 (EGM-2) with supplements (Lonza), serum-starved (18 hr) in EGM-2+0.5% FBS, and treated with TNFα (10 ng / ml; Sigma) for up to 30 mins. Prior to transfection cells were grown in antibiotic free EGM-2.

[0134]TALEN Design

[0135]Software developed by the Bogdanove laboratory was used to identify TALEN candidate binding sites (Doyle et al., 2012). Left and right TALENs were designed to contain 18 full monomer repeats, which target a 20 bp sequence, where the first and last bases are specified by the thymine at the N terminus, and the 0.5 repeat, respectively. To facilitate FokI dimerization, the left and right TALEN target sites were chosen with a spacer of 16-19 bp. For the pDT TALEN vector, the SAMD4A left and right arms were cloned into the pBI_CMV1 bidirectional promoter vector (Clontech). The left TALEN was cloned into MCS1 of pBI_CMV1 (MluI and HindIII) a...

example 2

[0174]Disrupting the IL8 Enhancer Abrogates IL8 Expression

[0175]Recently, Hi-C studies revealed that the pro-inflammatory chemokines CC Chr.17 (CCL2, CCL7, CCL11) and CXC Chr.4 (IL8, CXCL1, CXCL3, CXCL2) are organized into TADs and engage in chromosomal contact.

[0176]Using intronic smFISH, we were able to show that the CXC chemokines are only induced following TNF induction. Furthermore, the smFISH foci of co-expressed CXC genes always co-localize.

[0177]Deeper bioinformatic analysis of both Hi-C and ChIA-PET data in the CC and CXC TADs identified a large cluster of transcriptional enhancers. At the 5′ end of the CXC TAD, we identified a putative ‘super-enhancer’ region, spanning ˜80 kb and forming extensive chromosomal contacts with the proinflammatory genes (unpublished data). Typically, such regions are densely occupied by chromatin regulators over tens of kb. Accordingly, this region is highly enriched for eRNA chromatin marks, H3K4me1 and H3K27Ac. Using the recently published ‘e...

example 3

[0179]Disrupting Intra- or Interchromosomal Contact in Multigene Complexes Abrogates the Transcription of Interacting Genes (See FIG. 52)

[0180]TNFα, a major proinflammatory cytokine, is a stimulus that induces the coordinated assembly of coregulated genes in NF-κB-dependent multigene complexes (Papantonis et al., 2012). SAMD4A, a ˜221 Kb gene on chromosome 14, is rapidly switched on by TNFα in primary human umbilical vein endothelial cells (HUVEC). 4C analysis reveals that prior to stimulation with TNFα, SAMD4A seldom interacts with other genes.

[0181]After activation by TNFα, SAMD4A interacts with multiple coregulated genes to form a multigene complex. TNFAIP2, a gene located on the same chromosome but ˜50 Mb downstream, and SLC6A5 on chromosome 11, are two well-characterized interacting partners of SAMD4A. siRNA approaches cannot be utilized to interrogate loop-mediated dynamics in the SAMD4A / TNFAIP2 / SLC6A5 multigene complex, as all three genes are activated by the same transcripti...

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Abstract

This invention relates to a single cell assay for determining the effect of chromosomal contact on the transcriptional activity of genes of interest in a cell and to methods of silencing gene expression in a cell by way of perturbing gene regulatory elements which are engaged in chromosomal contact.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a Divisional Application of U.S. patent application Ser. No. 14 / 916,338, filed Mar. 3, 2016, which is a § 371 National Stage Application of PCT / IB2014 / 064259, filed 4 Sep. 2014 which claims priority to ZA 2013 / 06649, filed 4 Sep. 2013. Each of these applications is incorporated by reference in its entiretyREFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE (.txt)[0002]Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (see MPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-compliant text file (entitled “3000012-002001_Sequence_Listing_ST25.txt” created on 15 Nov. 2018, and 79,384 bytes in size) is submitted concurrently with the instant application, and the entire contents of the Sequence Listing are incorporated herein by reference.BACKGROUNDField of the InventionDescription of Related Art[0003]This invention relates to a single cell assay for determining the effect of chrom...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/01C12Q1/6876C12N15/63C12N9/22C12Q1/6841C12N15/11
CPCC12N15/01C12Q1/6876C12N15/63C12Q2600/158C12Q1/6841C12Q2600/136C12N9/22C12Q1/34
Inventor FANUCCHI, STEPHANIESHIBAYAMA, YOUTAROBURD, SHAUNMHLANGA, MUSA M.
Owner CSIR
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