Sequencing and analysis of MC3R gene using CRISPR/CAS9 and nanopore technologies

Debelost predstavlja velik javnozdravstveni problem in je pri otrocih povezana z razvojem bolezni kasneje v življenju. Nagnjenost k razvoju debelosti ni odvisna samo od okolja, ampak je preplet genetskih, okoljskih, vedenjskih in socialnih dejavnikov. Monogenska debelost je najpogosteje posledica genetskih sprememb v genih leptin-melanokortinske poti. V to signalno pot spada tudi gen za melanokortinski receptor 3 (gen MC3R), ki s svojim delovanjem zavira vnos hrane in pospešuje porabo energije. Namen magistrskega dela je bil pokazati, da lahko s pomočjo tehnologij CRISPR/Cas9 in sekvenciranja z nanoporami pri dveh skupinah preiskovancev določimo iskane spremembe, dosegamo primerne obogatitve regij in določimo metilacijski profil gena MC3R. Želeli smo pokazati, da je CRISPR/Cas9 učinkovit sistem za obogatitev tarčne regije genoma in da s sekvenciranjem z nanoporo lahko zaznamo že potrjeno spremembo v genu MC3R. Analizo smo izvedli na dveh skupinah preiskovancev. V prvi skupini je bilo šest preiskovancev s potrjeno genetsko spremembo, v drugi pa šest preiskovancev brez iskane genetske spremembe. Pridobljene podatke smo primerjali s podatki, pridobljenimi s sekvenciranjem naslednje generacije, ki temelji na branju kratkih fragmentov, in opredelili občutljivost in specifičnost sekvenciranja z nanoporo. Tehnologiji CRISPR/Cas9 in sekvenciranje z nanoporo sta nam tudi omogočili, da smo pri vseh preiskovancih določili metilacijski profil analiziranega genomskega odseka in poskušali najti razlike med skupinama. Rezultate smo pridobili skozi proces bioinformatske analize z namenskimi analitskimi orodji za podatke, pridobljene s sekvenciranjem na nanopori. Pri vseh preiskovancih v prvi skupini smo dokazali prisotnost znane spremembe. Po primerjavi z rezultati sekvenciranja naslednje generacije, ki temelji na branju kratkih fragmentov, smo ugotovili, da so rezultati sekvenciranja z nanoporo manj specifični, saj smo kot rezultat dobili več sprememb, kot je bilo pričakovano. Pri metilacijskem profilu je bil signal skozi celoten gen podoben, pri tem med preiskovanci in med skupinama nismo opazili razlik. Prišli smo do zaključka, da je s kombinacijo metod CRISPR/Cas9 in sekvenciranjem z nanoporo iskanje sprememb in določanje profila metilacije učinkovito.Obesity is a serious medical conditionchildhood 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 genegroup 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