Title Developing cas12j2 for programmable genome editing
Translation of Title Cas12j2 kūrimas programuojamam genomo redagavimui.
Authors Shah, Khushboo
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Pages 67
Keywords [eng] CRISPR-Cas, Cas12j2, Genome editing, DNA cleavage kinetics, NHEJ repair pathway, Indels, HEK293T cells, electroporation.
Abstract [eng] Clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized genome editing by allowing researchers to edit genomes with high precision in living organisms. There are different tool types in CRISPR technology; among them, nuclease-based CRISPR technology, specifically CRISPR-Cas, is one of the most popular and widely used. CRISPR-Cas is a natural adaptive immune system found in bacteria and archaea. Cas nuclease is directed by the single guide RNA to identify and cleaving target DNA sequence at the protospacer adjacent motif. Recently discovered Cas12j, a member of the Type V CRISPR-Cas nuclease family, has become a new focus of interest due to its structural and functional characteristics. It is a compact nuclease (700-800 kDa) with all the potential functions of a programmable genome-editing tool. In this study, we aimed to develop and validate Cas12j2 as a programmable genome-editing tool by producing and characterizing Cas12j2. We assessed the DNA cleavage activity of Cas12j2 and evaluated its potential for targeted DNA cleavage in human embryonic kidney (HEK293T) cells. Cas12j cleavage induces double-strand breaks with staggered cuts (sticky ends) at 5’ overhangs. These are primarily repaired by the error-prone non-homologous end joining (NHEJ) repair pathway, which generates insertions and deletions (indels) at the target site. Indels play a key role in gene disruption and functional studies. However, there is very limited information on Cas12j cleavage patterns mediated by the NHEJ repair system and the formation of indels. In this study, we aim to provide insights into the patterns of Cas12j2 indel formation to better understand editing precision. Findings suggest that Cas12j2-NLS can be successfully expressed in E.coli with high stability. The produced Cas12j2-NLS was approximately 50% enzymatically active. Cas12j2-NLS exhibits slower DNA cleavage kinetics compared to Cas9 and Cas12a. Successful optimization of the homemade Lonza AmaxaV buffer and electroporation conditions in HEK293T cells resulted in 30% of editing efficiency at the HPRT1 gene.
Dissertation Institution Vilniaus universitetas.
Type Master thesis
Language English
Publication date 2026