Uporaba metode CRISPR-Cas9 za zamenjavo posamičnega nukleotida in njen vpliv na genom kvasovke Saccharomyces cerevisiae

Doberšek, Katja (author), Petrovič, Uroš (mentor)

CRISPR-Cas9 je metoda za urejanje genoma, ki nam omogoča spreminjanje poljubnega zaporedja v genomu različnih organizmov. Deluje na osnovi sistematičnega uvajanja dvojnih prelomov DNA v izbrano zaporedje in uporabi celici lastnih popravljalnih mehanizmov za njihovo popravljanje na osnovi zaporedja DNA, ki ga v celico vnesemo sami. V kvasovki Saccharomyces cerevisiae se dvojni prelomi DNA lahko popravijo na dva načina. Prvi je homologna rekombinacija (HR), ki velja za prevladujočega, drugi pa je povezovanje nehomolognih koncev (NHEJ). Posledica NHEJ so pogosto indeli, torej krajše insercije in delecije, ter zamenjave nukleotidov na mestu dvojnega preloma, ki nastanejo zaradi procesiranja koncev. Da bi razvili metodo, s katero bomo lahko v genomu kvasovke na enostaven način zamenjali posamičen nukleotid s čim manj netarčnimi učinki delovanja encima Cas9, smo poskušali čim bolje okarakterizirati neželene učinke, ki nastanejo pri zamenjavi posamičnega nukleotida s CRISPR-Cas9. Opazovali smo vpliv delecije gena NEJ1, ki spodbuja NHEJ, vrste matrične DNA, ki jo uporabimo kot osnovo za popravljanje dvojnih prelomov po delovanju Cas9 in vpliv izgube komponent sistema CRISPR-Cas9 na učinkovitost delovanja sistema in nastanek indelov. Indele in druge netarčne učinke smo okarakterizirali tako na ravni posameznega tarčnega lokusa (v genu IXR1) kot na ravni celotnega genoma. Za to smo uporabili metode določanja nukleotidnega zaporedja naslednje generacije: sekvenciranje amplikonov in sekvenciranje celotnega genoma. Na osnovi sekvenciranja amplikonov in analize tako pridobljenih rezultatov smo opisali vpliv CRISPR-Cas9 na zaporedje tarčnega mesta na genomu oziroma v okolici dvojnega preloma DNA, če celica ne vsebuje matrice za HR. S sekvenciranjem celotnega genoma smo opisali še indele in zamenjave nukleotidov, ki so se pojavljali drugod v genomu in pri sekvenciranju amplikonov v analizo niso bili zajeti. Ugotovili smo, da se po delovanju sistema CRISPR-Cas9 v okolici tarčnega mesta na genomu, če ga ne spremenimo s HR z ustrezno matrično DNA, indeli skoraj vedno pojavijo in da njihov nastanek verjetno ni naključen.

• Language: Slovenian
• Keywords: kvasovka, CRISPR-Cas9, indeli, SNP, sekvenciranje amplikonov, sekvenciranje genomov
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-140892
• COBISS.SI-ID: 131912963
• Publication date in RUL: 21.09.2022

Secondary language

• Language: English
• Title: EVALUATION OF CRISPR-Cas9 SYSTEMS FOR SINGLE NUCLEOTIDE CHANGE AND ITS IMPACT ON THE GENOME OF YEAST Saccharomyces cerevisiae

Abstract

CRISPR-Cas9 is a genome manipulation method that allows us to modify a specific sequence in the genome of different organisms. It is used to systematically introduce double-strand DNA breaks at a selected target sequence in the genome and relies on cell's own repair mechanisms to correct them according to the DNA template that we introduce into the cell. In the yeast Saccharomyces cerevisiae, double-strand DNA breaks can be repaired in two ways. The first one is homologous recombination (HR), which is considered to be dominant over non-homologous end joining (NHEJ). NHEJ often results in indels and nucleotide substitutions at the site of the double-strand break. In order to develop a method to easily and efficiently replace a single nucleotide in the yeast genome with minimal off-target effects, we tried to characterise as good as possible the unwanted effects that arise when a single nucleotide is replaced with CRISPR-Cas9. We characterized the impact of the deletion of the NHEJ-promoting gene NEJ1, the type of DNA template used for double-strand break repair and the impact of system components loss after the CRISPR-Cas9 activity on the efficiency of the system itself and the formation of indels. Indels and other off-target effects were characterised on the level of the individual target locus (IXR1) and on the whole genome level. For this, we used next-generation sequencing methods, namely amplicon sequencing and whole genome sequencing. Using amplicon sequencing we characterised the effect of CRISPR-Cas9 on the sequence of the Cas9 target site and in the proximity of the DNA double-strand break if the cell did not contain the matrix for HR. We also used whole genome sequencing to characterise indels and nucleotide substitutions that occurred elsewhere in the genome and were not included in the amplicon sequencing analysis. We found that after CRISPR-Cas9 action, indels almost always appear in the vicinity of the target site on the genome if the appropriate HR template is not provided, and that their appearance is most likely not random.

• Keywords: yeast, CRISPR-Cas9, indel, SNP, amplicon sequencing, genome sequencing