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Author
Vila Alarcón, Adrià
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Abstract
Introduction: Disturbed fibroblast growth factor receptor 1 (FGFR1) structure and signaling is linked to the appearance of many types of pathologies, here emphasizing those of the brain, in particular low-grade neuroepithelial tumors (LGNETs) which are benign. Aiming to study the molecular mechanisms underlying FGFR1-driven LGNETs formation and progression, the design of CRISPR/Cas9-based FGFR1-knockout (KO) cell lines has been addressed. For that, assembly of a guide RNA (gRNA) targeting FGFR1 early exons and an endonuclease Cas9 generating double-strand breaks (DSBs) for insertions and deletions (INDELs) non-homologous end joining (NHEJ)-mediated to occur was required.
Results: After 65% and 20% lipofection efficiency of the gene editing machinery for human embryonic kidney (HEK293) and human oligodendroglioma (HOG) cell lines respectively; sorted cells through fluorescence activating cell sorting (FACS) were deposited as single cells in 96 well plates. Being each well a colony of clones, a total of 196 and 111 HEK293 and HOG clones, respectively, were screened first from higher-throughput and lower-selectivity techniques to the reverse at the end (1st: KO-PCR; 2nd: Western blot and 3rd: Sanger sequencing). Despite not having found any kind of FGFR1-KO clone, neither homozygous nor heterozygous, there are remaining candidates which have not gone yet through sequencing due to lack of time.
Conclusions: New gRNAs with higher edition efficiency should have been found during in vivo T7 endonuclease I (T7EI) validation assays prior screening. Nonetheless, current gRNAs were able to guide Cas9 to target loci in in vitro assays. No homozygous FGFR1-KO clone has been identified and further analysis is needed to evaluate the presence of heterozygous clones.
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