Genome-wide CRISPR-Cas9–based screening has identified essential regulators and major impediments of human primed to naïve pluripotent stem cell reprogramming. Researchers found that factors essential for cell state change do not typically undergo changes at the level of gene expression but rather are “repurposed with new functions.” The team also established that the variant Polycomb complex PRC1.3 and PRDM14 jointly repress developmental and gene regulatory factors to ensure naïve cell reprogramming.
The work was carried out by researchers at the Wellcome–MRC Cambridge Stem Cell Institute, the Epigenetics Programme at Babraham Institute in Cambridge, and collaborators. Their findings were published in Science this month.
They write that “The results describe the first genome-wide CRISPR-Cas9–based functional screen in human cell reprogramming and provide an important new dataset that can be mined by the scientific community to understand the processes controlling human pluripotent cell identity.”
Pluripotency is the ability of individual cells to give rise to all the tissue lineages of a mature organism. It arises during the epiblast phase of preimplantation embryos and lasts for two weeks until the postimplantation embryo gastrulates, when lineages are specified.
During this period, pluripotent cells isolated from embryos give rise to unspecialized human pluripotent stem cells (PSCs) that retain their developmental potential and characteristics. Pluripotency is can also be acquired when somatic cells are reprogrammed to become induced PSCs (iPSCs), creating a cell type that is largely indistinguishable from embryo-derived PSCs.
PSCs occur in two main states—naïve and primed. These states are functionally distinct, but both can self-renew and undergo multilineage. The authors write that, “Naïve PSCs largely recapitulate the transcriptome, epigenome, and differentiation potential of preimplantation embryos, and primed PSCs are similar to early postimplantation embryos.” As a result, naïve PSCs have sought-after properties, including the ability to generate extraembryonic cells and entire blastocyst-like structures, among other things.
This team aimed to define the genes that regulate the reprogramming of primed PSCs into a naïve state using a genome-wide CRISPR-Cas9–based screen.
First, they integrated the Cas9 coding sequence under the control of a CAG promoter into the safe harbor AAVS1locus in primed PSCs and confirmed that this modified cell line could reprogram to a naïve state with the expected proportion of naïve cells.
The Cas9-expressing primed PSCs were then transduced with an optimized human v3 CRISPR-based loss-of-function mutant library.
Genomic DNA was extracted from the two flow-sorted cell populations, and the amount of each gRNA was measured by high-throughput sequencing. The authors note that, “RNA sequencing (RNA-seq) libraries were also prepared from the same samples, and as expected, the transcriptional profiles of the two cell populations showed a strong correlation with previous naïve cell reprogramming experiments.”
The screen led the team to identify a new role for the noncanonical Polycomb repressive complex PRC1.3 in naïve PSC reprogramming. Expanding upon this finding, they looked further into how PRC1.3 and PRDM14 transcriptionally repress a set of developmental, chromatin, and signaling regulators to cause a failure to reprogram.
They note that, “It is likely that not all PRC1.3 target genes have detrimental effects, but having identified this gene set, these candidates can be examined in further studies as potential disruptors of cell reprogramming.”