Human iPS Cells

A Reference Parental Line

The parental cell line used for the generation of this catalog's gene-edited variants is the KOLF2.1J cell line. This cell line was selected based on a variety of characteristics including its genomic sequence, genome stability in culture, cellular characteristics (i.e., pluripotency) and amenability to gene editing protocols. The KOLF2.1J cell line was derived from the HPSI0114i-kolf_2 line through extensive molecular and cellular characterization efforts, including the correction of a pathogenic mutation in ARID2.

Single Nucleotide Variants

Our panel of SNVs was selected based on coding changes from inherited ADRD identified in the literature based on linkage and segregation of variants in families. In total, 134 variants spread across 73 genes were chosen to be engineered into the KOLF2.1J parental cell line. Each CRISPR-edited heterozygous and homozygous SNV cell line has been characterized through targeted sequence analysis (PCR-Sanger) and quality controlled using karyotype, genomic wide copy number variant and molecular pathogen analyses. We offer both heterozygous and homozygous cell lines to enable assessments of allelic dosage effects.

Non-Desired Mutations: CRISPR-based editing procedures can inadvertently produce mutations outside the sequence region used in the characterization process. These are frequently referred to as “off-target” effects. These mutations are typically distant from the region being targeted, most often residing on other chromosomes. In contrast, the same process can yield mutations in the region of interest that can go undetected by the targeted PCR-Sanger sequencing process. Even current “next-generation” sequencing technologies and their associated computational algorithms can fail to identify these aberrant “on-target” mutations. Most “off-target” effects have very similar sequences when compared to the desired targeted sequence and can be mitigated in the design strategy. In contrast, the “on-target” aberrant mutations are less predictable. These on- and off-target mutations can lead researchers to inappropriately assign functional significance to the engineered SNV, even when parental controls are used.

Rescue Mutation: “Hidden” undesired mutations can lead a user of an engineered cell line to misattribute phenotype causation to the declared SNV when the true cause is the uncharacterized mutation elsewhere in the engineered cell line genome. To reduce the risk that a researcher is misled by one or more of the above non-desired mutations in the SNV-carrying cell line, we have created precise, efficient controls. The SNV mutation-carrying cell line has undergone another round of gene engineering to revert the gene back to a 'wild type' status. We refer to these cell lines as “revertants” and they represent a high-value control for your in vitro experiments. If one detects a phenotype in the SNV-containing cell line compared to the parental control but the “revertant” continues to produce the same phenotype as the SNV cell line, the phenotype is likely caused by one, or several, undesired mutations.