Researchers have conducted a study, published in Molecular Cell, to investigate what features influence how well CRISPR activation works for different sets of genes to help “write the rulebook on how to effectively use CRISPR activation technology”.
CRISPR activation – what is it?
CRISPR activation (CRISPRa) allows scientists to overexpress certain genes through epigenetic modification. This technique uses a protein called Cas9, which is designed to bind to specific sequences of DNA in the genome. The Cas9 protein is modified to be catalytically inactive and to recruit other proteins that can activate or repress the targeted genes. This targeted manipulation of gene expression allows researchers to study the function of individual genes and regulatory elements. However, predicting the efficiency of CRISPRa at specific points in the genome remains challenging, which makes it difficult to reliably overexpress certain genes. Despite this limitation, CRISPRa has been widely used in functional genomics research and is a useful tool for understanding the role of specific genes in biological processes.
Gene activation roulette
The research team, from the Wellcome Sanger Institute (and collaborators), used a human stem cell line and integrated a minimal barcoded reporter gene at thousands of points along the genome. The reporter gene consisted of a synthetic core promoter and a Venus fluorescent protein with a randomized 17-nucleotide barcode in the 3’UTR.
They then activated the marker gene with CRISPRa and tested how efficiently the genes were activated. The human stem cell line was also induced to differentiate into neurons, which allowed the team to assess CRISPRa efficiency in different cell types. Moreover, the differentiation of induced pluripotent stem cells (iPSCs) to neurons also provided an ideal platform to assess how the environment surrounding the genome and changes in basal gene expression affect CRISPRa efficacy.
It’s all about the chromatin
The study tested two types of CRISPRa constructs, dCas9-VPR, which was a dead Cas9 fused to an artificial transcription activator called VPR, and dCas9-p300, a dead Cas9 fused to the histone acetyltransferase p300. Surprisingly, they found that the two CRISPRa constructs behaved very differently. dCas9-VPR was able to activate most barcoded reporter genes, independent of chromatin status, whereas dCas9-p300 could not.
The team found that whilst most genes can be activated by dCas9-VPR, not every cell responded to the same extent. So the team decided to investigate additional chromatin features and how that affects CRISPRa efficiency. The location of a gene within the chromatin structure, and the presence of certain histone modifications, can impact the level of gene activation.
Bivalent genes, which are genes that have both activating and repressing histone modifications in their regulatory regions, were found to be particularly sensitive to dCas9-VPR. On the other hand, genes that were “heterochromatin”, for example those that contained a histone mark called H3K9me3, a repressing regulator which shields the gene from activation, showed variation in response. The study also tested whether these rules can be applied to endogenous loci using a parallel single-cell-based CRISPRa assay and demonstrated that CRISPRa could elicit strong activation, which corresponds to the top 20% of endogenous gene expression levels.
A CRISPR activation “rulebook”
Senior author of the study, Andrew Bassett summed up the study by saying “CRISPR activation is a widely used, and incredibly valuable technique when it comes to genome editing, and our research aims to support those using it and help them get the most out of their experiments. By investigating the factors that impact CRISPRa efficiency in a systematic way, we have created a set of rules that show where it is most or least effective and deepened our understanding of the factors involved.”
One notable finding from the study was that the sensitivity of bivalent genes to dCas9-VPR suggest that CRISPRa may be able to manipulate stem cell differentiation. Although this requires further studies to investigate this potential use, as Qianxin Wu, first author of the study notes “Our research has established a system for reporting the effectiveness of CRISPR activation in stem cells, allowing us to gain a better understanding of how CRISPRa works in multiple cell states. We also showed that CRISPR gene activation is powerful enough to induce stem cells to differentiate into other cell states. This suggests that CRISPRa screens can be used to search for genes involved in cellular processes or to generate more accurate models of cell types in the body, aiding research into genetic diseases and regenerative medicine.”