Telomeres are repetitive nucleoprotein structures at the ends of linear chromosomes which protect essential coding DNA from degradation due to the end replication problem. In this, they act as a molecular clock limiting cellular replicative lifespan. Thus, telomere length regulation plays a complex role in human health. Aberrantly short telomeres in stem and progenitor cell populations drive premature aging and tissue failure disorders. Conversely, excessively elongated telomeres cause heritable cancer predisposition syndromes. Therefore, careful regulation of telomere length is essential to human health.

Shelterin, the heart of telomere length regulation, is a six-member complex of proteins which binds to telomeric repeats, suppressing the activation of DNA damage response machinery and modulating the access of telomerase to telomere ends. Two Shelterin proteins with clear clinical ties are POT1 and TIN2, wherein germline mutations have been linked to both cancer and telomere biology disorders. To provide a functional map of essential residues in each protein and classify clinical variants of unknown significance, we performed CRISPR/Cas9 mediated deep scanning mutagenesis in locally haploid human embryonic stem cells. We introduced synonymous mutations, alanine substitutions, single codon deletions, premature stop codons, and clinical variants of unknown significance across the gene body of both POT1 and TIN2. Using this method, we identified a subclass of cancer-associated POT1 mutations which actively promote telomere elongation and mapped the TIN2 truncation point wherein mutations drive telomere shortening rather than telomere elongation.