Understanding the factors that impact CRISPRa efficiency

Investigators from the Wellcome Sanger Institute and their partners utilized human stem cells and neurons to explore what attributes impact how well CRISPR activation works for different sets of genes.

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The study, published on March 13th, 2023, in the journal Molecular Cell, describes the rules that determine how much genes respond to CRISPR activation, guaranteeing that future research can be crafted as effectively as possible.

CRISPR activation (CRISPRa) is a type of CRISPR gene editing in which specific genes are overexpressed. Although this technique is widely used, estimating its effectiveness when directed at specific points in the genome can be difficult, making it difficult to reliably overexpress specific genes.

The Wellcome Sanger Institute and collaborators conducted new research in which they 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 researchers to learn about CRISPRa efficiency in various cell types.

The researchers noted several factors that influence CRISPRa efficiencies, such as expression level, cell state, chromatin status, and gene location. They discovered that CRISPRa can strongly activate bivalent genes, which are important developmental regulator genes with both repressing and activating marks in the same region.

They also noticed that CRISPRa could achieve the same levels of overexpression required to cause significant changes in cell state and differentiation.

The researchers discovered that while CRISPRa can activate most genes, not every cell responds in the same way. Genes that encompass H3K9me3 repressing regulators, and thus are protected from activation, exhibited increased variation in response.

These findings show for the first time that CRISPRa is applicable across chromatin states and cell types, as well as the factors that influence gene activation and how easy it is to replicate the effects. Understanding these factors is critical for the design and analysis of CRISPRa screens, which are used to identify genes involved in genetic diseases.

More research is needed to expand on these rules and determine whether different CRISPRa or CRISPR interference techniques behave similarly.

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.

Dr. Qianxin Wu, Study First Author, Wellcome Sanger Institute

Dr. Qianxin Wu adds, “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.”

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.”

Dr. Andrew Bassett, Study Senior Author, Wellcome Sanger Institute

Source:
Journal reference:

Wu, Q., et al. (2023). Massively parallel characterization of CRISPR activator efficacy in human induced pluripotent stem cells and neurons. Molecular Cell. doi.org/10.1016/j.molcel.2023.02.011.

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