Generation of Knockout Human iPSCs to Investigate Genes Associated with Telomere Length

Telomeres are repetitive sequences located at the ends of human chromosomes. During DNA replication, DNA polymerase is unable to fully replicate telomeric DNA causing a progressive reduction in telomere length with each cell division. Consequently, telomeres shorten with age and when telomere length reaches a critical length the cell becomes dysfunctional or senescent. Increasing numbers of senescent cells results in reduced organ function and telomere length has been associated with age-associated diseases including cardiovascular disease and cancer. Telomere length is highly heritable and genome-wide association studies have identified several loci that associate with telomere length. Interestingly, two of these loci do not contain genes with known roles in telomere maintenance. In this study, CRISPR/Cas9 genome editing was used to knockout telomere length associated genes in human induced pluripotent stem cells (hiPSCs) with the ultimate aim of definitively linking a novel telomere length associated gene to telomere maintenance. CRISPR/Cas9 was used to create hiPSCs with no telomerase activity by knocking out TERT, which encodes the catalytic subunit of telomerase. Telomere length was maintained in control iPSCs, but reduced by approximately 1% per day during extended culture of TERT knockout hiPSCs. Flow cytometric analysis of mutant hiPSCs revealed that they continued to express markers of pluripotency but had increased expression of a differentiation marker. Next, CRISPR/Cas9 was used to generate mutations in the candidate telomere maintenance genes ACYP2 and TSPYL6, however, no difference in telomere length was observed after extended culture of hiPSCs carrying mutations in either gene. In conclusion, CRISPR/Cas9 genome editing was successfully used to generate mutant hiPSCs for TERT, which resulted in telomere shortening and for two candidate telomere maintenance genes, which had no effect on telomere length. Further analysis will be required to determine which gene mediates the association with telomere length at the ACYP2 locus.