NEW YORK (GenomeWeb) – Researchers at the Wellcome Trust Sanger Institute and the University of Cambridge have optimized RNAi- and CRISPR/Cas9-based platforms for loss of function studies in human pluripotent stem cells.
Specifically, the researchers created and validated platforms using short hairpin RNAs and CRISPR/Cas9 constructs to knock down and knock out genes in hPSCs and cells differentiated from them.
"Most other systems only work in one specific cell type," senior author Ludovic Vallier, who is affiliated with both the Sanger Institute and University of Cambridge, told GenomeWeb. "Ours is good in all the different cell types that can be derived from human pluripotent stem cells."
On Tuesday, the authors described their plasmid-based, single-step optimized inducible gene knockdown (sOPTiKD) or knockout (sOPTiKO) platforms in Development.
Vallier stressed two other features of the platforms: not only can they be used in different cell types, they're useful in different stages of development and they allow multiplex gene knockdown or knockout, such as an entire family of genes.
"This is a clever application of gene targeting and inducible transgene technologies that enables more efficient knockdown and knockout of specific genes to investigate their roles in human pluripotent stem cell development," Jeanne Loring of the Scripps Research Institute told GenomeWeb in an email. "Data acquired by these and similar methods will be valuable for improving our understanding of human development and disease, especially inherited diseases."
Vallier said that his interest in this subject goes back many years and that his group began working on optimizing these platforms for work on genes involved in disease about two years ago. He was trying to look at gene expression in different stages of oncogene development, but couldn't see it in fully differentiated cells.
In the paper, the authors noted that shRNA in hPSCs is plagued by several problems, including loose control of shRNA expression in general and transgene silencing inhibiting promoters in both hPSCs and differentiated cells.
For sOPTiKD, the researchers found multiple safe harbor loci for different components of the tetracycline promoter system. This TET-ON system was based on dual genomic safe harbor targeting, with codon optimization for the tetracycline-sensitive repressor protein (tetR). They validated the system in human embryonic stem cells, hPSCs, and hPSC-differentiated progeny cells, inducing knockdown of EGFP protein.
They also developed a knockout system using CRISPR/Cas9. "Current inducible CRISPR/Cas9 methods rely on conditional overexpression of Cas9 in the presence of a constitutively expressed gRNA," which also relies on a TET-ON system, the authors wrote. "Although this TET-ON platform has been successfully applied to certain human cell types, we observed that this inducible system is silenced during hPSC differentiation into multiple lineages," including cardiomyocytes, hepatocytes, and smooth muscle cells.
This sOPTiKO system opens up new lanes of research in developmental biology and cancer.
"Before, if we knocked out a gene, we could only see what effect this had at the very first step," Vallier said. "By allowing the gene to operate during the cell's development and then knocking it out with sOPTiKO at a later developmental step, we can investigate exactly what it is doing at that stage."
It also opens up new cell types for investigation. "We can get hepatocytes from patients easily but they don't have a stable phenotype," he said. "Doing gene knockout in those cells is impossible." But the new platform will now work in hPSCs differentiated into hepatocytes. "It lets us do functional experiments that would be impossible otherwise," he said.
In cancer research, investigators can now target entire gene families, like cell cycle regulators. And not only can they look at oncogenes in one cell type, they can see the function of the same gene or gene family in multiple tissue types in parallel.
Vallier said he plans to submit plasmids to the Addgene repository in about a month, but all reagents are freely available directly from his lab.