Detection of genomic changes induced by CRISPR/Cas9 in Chinese hamster ovarium cells using high resolution melting curve analysis

Metoda CRISPR/Cas9 je najbolj uporabljena metoda tarčnega spreminjanja genoma. Pregledovanje in identifikacija CRISPR/Cas9 spremenjenih celičnih klonov je drag ter časovno in delovno zahteven postopek. Metoda analize talilne krivulje visoke ločljivosti (HRM) je hitra in stroškovno ugodna metoda detekcije CRISPR/Cas9 induciranih DNA sprememb. Z izbranimi reagenti smo po izolaciji gDNA, izolat gDNA iz ovarijskih celic kitajskega hrčka (CHO) 5-krat redčili, saj smo ugotovili, da ta redčitev omogoča najbolj ponovljive vrednosti Tm talilnih krivulj. Metoda HRM je uspešna ob uporabi vsaj 5000 celic CHO na vzorec. Z metodo HRM smo preverili prisotnost genetskih sprememb tarčnih regij v 105 klonih celic CHO. Vsak izmed klonov je bil tarčno spremenjen v enem od petih genov, na dveh mestih v genu hkrati. V nekaterih primerih smo pridobili talilne krivulje, ki zelo odstopajo od pričakovanih. Odstopajo predvsem rezultati tistih klonov, kjer smo zaradi velike oddaljenosti med tarčnima mestoma v genu, za pomnoževanje z reakcijo PCR uporabili dva para začetnih oligonukleotidov. Po izločitvi teh klonov iz analize rezultatov HRM ter njihovi določitvi kot genetsko spremenjene, smo primerjali rezultate metode HRM z rezultati metode naslednje generacije sekvenciranja (NGS). Genetske spremembe inducirane s sistemom CRISPR/Cas9 pri celicah CHO smo z metodo HRM določili s 95 % točnostjo in 96 % občutljivostjo glede na metodo NGS. Ob hkratni analizi dveh tarčnih mest v isti tarčni regiji z enim parom začetnih oligonukleotidov, smo genetske spremembe določili s 100 % natančnostjo metode HRM.CRISPR/Cas9 method is the most used method to induce targeted genomic changes. Screening and identification of CRISPR/Cas9 induced genomic changes in cell clones are expensive, time-consuming and labour intensive approaches. A high resolution melting (HRM) method enables cost-effective and rapid detection of CRISPR/Cas9 induced changes of DNA. After gDNA isolation of Chinese hamster ovarium (CHO) cells with specific reagents, we discovered that 5-fold dilution of isolated gDNA is the most optimal, based on the reproducibility of Tm value. Minimal number of cells for successful HRM method was specified, being at least 5000 CHO cells per sample. Furthermore, genomic changes of 105 clones were screened by HRM method. Every clone was targeted at two target sites inside one of five genes by CRISPR/Cas9 system. However, besides regular HRM results of five target regions, we obtained some irregular melting curves (outliers) of specific clones. Due to a considerable distance between target sites inside specific genes, we amplified those target sites with two different primer pairs. After exclusion of clones with irregular melting curves and determined them as genetic changed, we compared results of HRM method with results of next-generation sequencing method (NGS). Our results showed 95 % accuracy and 96 % sensitivity of the HRM method. If we consider clones, whose both target sites were amplified only with one pair of primer, we can conclude that we detected genetic changes with 100 % accuracy