Targeted DNA methylation of MGAT3 gene promoter using a CRISPR‐Cas9 tool

Josipović, Goran (2016) Targeted DNA methylation of MGAT3 gene promoter using a CRISPR‐Cas9 tool. Diploma thesis, Faculty of Science > Department of Biology.

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Abstract

The  CRISPR‐Cas9  system  allows  targeted  modifications  and  manipulation  of  any  genomic  loci in different cell lines. The basic principle of the method is based on the directing of non‐specific nuclease Cas9 to a specific area in the genome using sgRNA molecule, in which 20 nucleotides (nt) at 5' end is crucial in recognizing the complementary sequence. The advantage of the CRISPR‐Cas9 system  is  in  its  simple  manipulation  and  simultaneous  action  of several CRISPR‐Cas9 complexes  guided by different sgRNA molecules. System modifications include the fusion of catalytic domain of effector protein to the inactive nuclease Cas9 (dCas9), thus enabling editing of the genome and epigenome  without  inducing  double  stranded  break  in  the  DNA  molecule.  The  modification  of  CRISPR‐Cas9 system, resulted by fusing the DNA sequence encoding the catalytic domain of de novo  DNA  methyltransferase  3A  (DNMT3A)  with  the  DNA  sequence  encoding the inactive  nuclease dCas9 via a flexible Gly4Ser linker, enables targeted methylation of DNA in the genome. The  aim  of this  study was to  construct  epigenetic editing CRISPR‐Cas9 tool for targeted  DNA  methylation of MGAT3 gene promoter close to transcription start site (TSS) and to determine the effect of the target region methylation on the regulation of MGAT3 gene expression. Constructs for targeted methylation were successfully assembled by cloning sequences of sgRNA molecules into plasmids. For plasmid constructs cloning the bacterial strain E. coli XL10‐Gold was  used.  The  plasmid  constructs  were  successfully  transfected  into the BG1 cell  line.  After  the  isolation of total RNA, reverse transcription and real‐time polymerase chain reaction (qPCR), the results show no statistically significant reduction in MGAT3 gene expression in the cells transfected with plasmid constructs that contained active DNMT3A, nor in the control samples. Pyrosequencing was  not  done  to  prove  if  the  targeted  DNA  methylation  was  induced  due  to  the  inability  of  multiplying bisulfite converted DNA from BG1 cells, so the results of qPCR analysis cannot be accepted with confidence.

Item Type: Thesis (Diploma thesis)
Keywords: genome and epigenome editing, CpG island, DNMT3A, dCas9, BG1 cells, expression
Supervisor: Vojta, Aleksandar and Dobrinić, Paula
Date: 2016
Number of Pages: 61
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Grozdana Sirotic
Date Deposited: 24 Nov 2016 07:40
Last Modified: 24 Nov 2016 07:40
URI: http://digre.pmf.unizg.hr/id/eprint/5340

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