Obesity is a serious medical condition; childhood obesity can lead to severe complications later in life. It is a multifactorial disease depending on environmental, genetic, behavioural, and social factors. Monogenic obesity is often caused by genetic variants in genes involved in the leptin-melanocortin pathway. The pathway also includes the melanocortin 3 receptor (MC3R), which is encoded by the melanocortin 3 receptor gene. Its function is to decrease food intake and elevate energy utilization.
The purpose of this project was to show that CRISPR/Cas9 and nanopore sequencing technologies are efficient for DNA enrichment, genetic variants discovery, and determining the methylation profile of samples. We wanted to show, that CRISPR/Cas9 provides efficient target region DNA enrichment and nanopore sequencing enable us to determine known genetic variant in the MC3R gene. Samples were separated into two groups: group 1 – six samples with a known genetic variant in the MC3R gene; group 2 – six samples without the known genetic variant in group 1. Data about genetic variants collected with nanopore sequencing were compared with short-read next-generation sequencing data about genetic variants to determine the specificity and sensitivity of nanopore sequencing. Both CRISPR/Cas9 and nanopore sequencing enabled us to determine the methylation profile of each sample as well.
Results were obtained with bioinformatic analysis using tools specifically designed to process data collected with nanopore sequencing. In group 1, all the samples had the genetic variant we were trying to identify. In both groups, we found more genetic variants with nanopore sequencing than with short-read next-generation sequencing, which was expected. The results of nanopore sequencing had lower specificity because we obtained more genetic variants with nanopore sequencing. The methylation profile was similar in all the samples and between both groups. We concluded that using CRISPR/Cas9 in combination with nanopore sequencing is an efficient way of finding genetic variants and determining the methylation profile.
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