Characterizing de novo variants in the CRISPR-Cas9 edited KOLF2.2J iPSC cell line


Alyssa Lima

Document Type


Publication Date

Summer 2023



JAX Location

In: Student Reports, Summer 2023, The Jackson Laboratory


Cell culture systems are often thought to be homogeneous models for biomedical research; however, cell lines can accumulate mutations over time to create heterogenous and genetically distinct cellular populations. Therefore, the genomic integrity of cell lines must be established to standardize the use of cell culture models for biomedical research. To investigate mutations in cell culture systems, we will profile genome-wide genetic variation in the KOLF2-C1 cell line along with two serial derivatives, KOLF2.1J and KOLF2.2J, containing CRISPR-Cas9 corrections of pathogenic mutations. This project proposes to determine if establishment of the fibroblast derived cell line or subsequent CRISPR-Cas9 gene editing and serial passaging has led to de novo variants and potential phenotypic effects within the KOLF2.1J and KOLF2.2J cell lines. We will utilize a multiple-caller approach and orthogonal techniques (multiple methods to perform the same task) to identify and verify single nucleotide variants (SNVs), indels (insertions or deletions < 50 nucleotides), and structural variants (mutations ≥ 50 nucleotides, SVs) across the KOLF2.2J line in comparison to the KOLF2.1J and KOLF2-C1 lines it was derived from, and will also investigate variants distinct between these three lines and the human genome reference sequence. Then, we will annotate the variants for potential effects on known genes and regulatory elements. By identifying and characterizing mutations within these three cell lines, we seek to validate the model as well as determine the genomic stability of induced pluripotent stem cells (iPSCs) which have undergone serial rounds of CRISPR-Cas9 editing.

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