Abstract
CRISPR/Cas9 technology enables efficient loss-of-function analysis of human genes using somatic cells. Studies of essential genes, however, require conditional knockout (KO) cells. Here, we describe the generation of inducible CRISPR KO human cell lines for the subunits of the telosome/shelterin complex, TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, which directly interact with telomeres or can bind to telomeres through association with other subunits. Homozygous inactivation of several subunits is lethal in mice, and most loss-of-function studies of human telomere regulators have relied on RNA interference-mediated gene knockdown, which suffers its own limitations. Our inducible CRISPR approach has allowed us to more expediently obtain large numbers of KO cells in which essential telomere regulators have been inactivated for biochemical and molecular studies. Our systematic analysis revealed functional differences between human and mouse telomeric proteins in DNA damage responses, telomere length and metabolic control, providing new insights into how human telomeres are maintained.
Highlights
In the past 20 years, we have gained tremendous insight into how the ends of mammalian chromosomes or telomeres are maintained and regulated
Much of our knowledge regarding the molecular and functional significance of mammalian telomeric proteins comes from studies using mouse knockout (KO) mouse embryonic fibroblast (MEF) cells, as genes are more readily targeted in mouse embryonic stem cells
Even with effective KD (480%) of TRF2, for example, we could only observe minor DNA damage responses (DDRs) at telomeres, rarely more severe phenotypes such as chromosome end-to-end fusions found in Trf2 KO MEF cells [50], suggesting that residual TRF2 proteins in the KD cells may have been sufficient to prevent severe and sustained telomere DNA damages
Summary
In the past 20 years, we have gained tremendous insight into how the ends of mammalian chromosomes or telomeres are maintained and regulated. Human telomeres are considerably shorter than those of laboratory mice and human has one POT1 gene, whereas mouse has two (Pot1a and Pot1b) Such disparities underscore the need for loss-of-function human cellular models. The limitations of RNAi knockdown (KD) and the fact that several key telomere regulators including TRF2 and TIN2 are essential genes have complicated data analysis and interpretation. Complete inactivation of these telomere regulatory genes in cells may cause cell death, precluding further detailed biochemical and molecular studies, especially experiments that require extended culturing and/or large numbers of cells
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