UK researchers, from the Wellcome Sanger Institute and collaborators, have used human stem cells and neurons to investigate what features influence how well CRISPR activation works for different sets of genes.
Published in Molecular Cell, the study explains the rules determining to what extent genes respond to CRISPR activation, ensuring that future research can be designed as efficiently as possible.
CRISPR activation (CRISPRa) is a type of CRISPR gene editing that is used to overexpress certain genes. Although this technique is broadly used, predicting its efficiency when aimed at certain points in the genome can be challenging, making it hard to reliably overexpress certain genes.
The new study integrated a marker gene at thousands of points in the genome of a human stem cell line, which was then activated with CRISPRa, to see where this was successful. The stem cell line used differentiates into neurons, allowing the team to also gather information on CRISPRa efficiency in different cell types.
Dr Andrew Bassett, senior author from the Wellcome Sanger Institute, commented: “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.”
Factors that impact CRISPRa efficiency
The team uncovered multiple features that impact CRISPRa efficiency, including expression level, chromatin status, cell state, and gene location. They found that bivalent genes, which are key developmental regulator genes that have both repressing and activating marks in the same region, can be robustly activated by CRISPRa.
They also discovered that CRISPRa could achieve the same overexpression levels that are necessary to drive significant changes in cell state and cause them to differentiate.
Whilst most genes can be activated by CRISPRa, not every cell responded to the same extent. For example, genes that contain H3K9me3 repressing regulators, and therefore are shielded from activation, showed greater variation in response.
“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,” said Dr Qianxin Wu, first author from the Wellcome Sanger Institute. “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.”
Further research is required to continue to add to these rules and to see whether different CRISPRa or CRISPR interference techniques behave in a similar way.