New research writes the rules on CRISPR activation
Researchers have started to map out the rules on how to use CRISPR activation techniques in the most effective way, assisting in the design of future studies.
Researchers, from the Wellcome Sanger Institute and collaborators, used human stem cells and neurons to investigate what features influence how well CRISPR activation works for different sets of genes.
The new 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.
This new study, from the Wellcome Sanger Institute and collaborators 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.
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.
The team found that 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.
These data demonstrate for the first time that CRISPRa is generally applicable across chromatin states and cell types, and highlights the factors that impact the degree of gene activation and how easy it is to reproduce the effects. Understanding these factors is important in the design and analysis of CRISPRa screens, which are used to look for genes involved in genetic diseases.
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.
https://www.sanger.ac.uk/news_item
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