Murdani Abdullah, MD1, Ari Fahrial Syam, MD, FACG1, Marcellus SImadibrata, MD, PhD, FACG1, Sofy Meilany2, Firda Annisa, MD1, Dimas Ramadhan2, Dadang Makmun, MD, FACG1, Abdul Aziz Rani, MD1
1Universitas Indonesia, Jakarta Pusat, Jakarta Raya, Indonesia; 2Virology and Cancer Pathobiology Research Center, Universitas Indonesia, Jakarta Pusat, Jakarta Raya, Indonesia
Introduction: The mismatch repair genes as MLH-1 facilitate the cells to detect and repair bases mismatch that oftenly occur during replications process. Mutations of MLH-1 genes may be inherited and increase the risk for the related person to develop severals tumors including colorectal cancer. The development of gene therapy in the recent decades arise ideas in developing experimental gene editing using CRISPR-Cas9 to the hereditary colorectal cancer patients primary cells. The objective of this study is to apply the CRISPR-Cas9 MLH-1 editing technology to primary cells of colorectal cancer primary cells from hereditary colorectal cancer patient stage 4.
The cells from patient biopsy with diagnosis colorectal cancer with mucinous type, the cells
were extracted using Collagenase Type I and cultured in Fibroblast Growth Medium for several passages. The cells were then plated in 96-well culture plate. For genome editing we use ribonucleoprotein (RNP) consisting of Alt-R S.p. Cas9 nuclease in complex with Alt-R CRISPR-Cas9 guide RNA. We design this sg RNA
targetted MLH-1 sequence using USCS genome browser. The cells then transfected with MLH-1 CRISPR-Cas9 using DMERIC reagent according to the manufacture protocols for 48 hours. After incubations, the cells were harvested. The DNA were isolated using TriZol reagent. Analysis of mutation
were using T7E1 enzyme and primers were checked afterwards.
Results: From our study we found that efficiency of introduction of complex Alt-R S.p. Cas9 nuclease in complex with Alt-R CRISPR-Cas9 guide RNA only 40 % from total cells, the analysis was performed using Tali Cytometri. For amplification and analysis of Nickase gene was using T7E1 for primary cells of colorectal cancer primary cellswe found that this gene was amplified. From this finding this methods might be alternative therapy for colorectal cancer.
Discussion: Colorectal cancer result from progressive accumulation of multiple genetic and epigenetic aberrations within cells. The progression from colorectal adenoma to carcinoma is caused by three major pathways: microsatellite instability, chromosomal instability and CpG island methylator phenotype. Until now in Indonesia we still cannot define which pathway are the most common. This study we try to find out response of MLH 1 gene editing using CRISPR Cas 9. From our result we found that MLH 1 gene were able to mutated by using T7E1 assay and it was confirmed using Sanger Sekuensing.
Citation: Murdani Abdullah, MD; Ari Fahrial Syam, MD, FACG; Marcellus SImadibrata, MD, PhD, FACG; Sofy Meilany; Firda Annisa, MD; Dimas Ramadhan; Dadang Makmun, MD, FACG; Abdul Aziz Rani, MD. P0121 - TARGETING MLH-1 GENE OF COLORECTAL CANCER CELL LINE DEVELOP FROM PATIENT OF CIPTO MANGUNKUSUMO NATIONAL HOSPITAL BY CRISPR CAS9 FOR GENOME EDITING. Program No. P0121. ACG 2019 Annual Scientific Meeting Abstracts. San Antonio, Texas: American College of Gastroenterology.