Novel CRISPR screen speeds discovery of early developmental regulators

Key points:

• A novel approach combining CRISPR-activation and single-cell analysis enables the systematic identification of key inducers of zygotic genome activation-like transcription in embryonic stem cells.
• The research combined a high-throughput novel experimental method with a computational approach based on multi-omics factor analysis.
• The approach identified novel factors that induce ZGA-like transcription, such as the transcription factor Patz1 and the chromatin remodeller Smarca5, as well as previously known regulators such as the DNA-binding protein Dppa2.

Epigenetic researchers at the Institute have been the first to use precise gene expression control and single-cell techniques to learn more about the ‘ignition spark’ at the very beginning of embryonic development. This process, called zygotic genome activation (ZGA), is a crucial developmental milestone and marks the start of the interpretation of the genomic information to create a new life. The novel high-throughput screening method identified new regulators of the genome activation process and gave insight into how newly identified and know regulators work together. The research is published in the journal Cell Systems today.  

The researchers first used CRISPR genome editing technology (CRISPR-activation) to drive the individual expression of 230 maternal epigenetic and transcriptional factors in mouse embryonic stem cells (ESCs). By combining this CRISPR activation with single cell RNA sequencing, the team were able to analyse the genes expressed as a result of the overexpression of each factor. This generated hundreds of thousands of readouts meaning that computational analysis was required to identify outcomes of relevance to zygotic genome activation. 

The team, bringing in expertise from Oliver Stegle from the EMBL-European Bioinformatics Institute, used a computational approach called multi-omics factor analysis (MOFA) to mine the single cell readouts and find factors that promoted a ZGA-like response. MOFA manages data in an unbiased way, and the authors interpreted this output to understand biological processes that explain the data, identifying a strong signature related to ZGA transcription. This use marked the first application of MOFA to single-cell CRISPR screening.

Dr Celia Alda-Catalinas, who developed this project as part of her PhD in the Reik lab and is the first author on the paper, said: “By combining high-throughput CRISPR screening with single-cell transcriptomics, we were able to generate a bank of data that allowed the identification of new regulators of ZGA-like transcription in embryonic stem cells. It’s been an exciting piece of work for two reasons: developing a new screening approach to allow us to perturb the system and analyse the outcome of the perturbations using a complex and rich read-out, and also to see that we were able to identify and validate relevant hits that can be followed-up in in vivo studies. The majority of our hits have not been previously linked to ZGA so these findings really help expand what we currently know about this fascinating developmental event.”

The MOFA analysis uncovered 24 epigenetic and transcriptional factors that induced a pattern of transcription that resembled ZGA. Further experimental work looking at nine of these factors validated both the combination of CRISPR-activation and single cell sequencing plus the use of MOFA to identify hits of interest.

Three particular factors were focused on in greater detail: Patz1 – a transcription factor, Dppa2 – already known to be key in ZGA, and Smarca5 – a chromatin remodeller.

Dr Mélanie Eckersley-Maslin, a BBSRC Discovery fellow in the Reik lab, said: ‘This work adds to what we know about zygotic genome activation and provides valuable insights into what is still a poorly understood aspect of biology. In addition, it gives us a way to learn more about the molecular mechanisms that orchestrate ZGA.”

Professor Wolf Reik, Epigenetics group leader and the Institute’s Acting Director, said: “The study of zygotic genome activation is growing into an exciting field but so far there has not been an approach to survey and identify its regulators at a systems level; this is precisely what our work establishes. We anticipate that this screening method could be used to investigate other transcriptional programmes and so is widely adaptable to multiple biological contexts.”

The work builds on the team’s recent work on zygotic genome activation including identifying two important proteins called Dppa 2 and Dppa4 and their role in zygotic genome activation.

Notes to Editors

Publication reference

Alda-Catalinas, C. et al.

A single-cell transcriptomics CRISPR-activation screen identifies epigenetic regulators of the zygotic genome activation programme.

Cell Systems. DOI: 10.1016/j.cels.2020.06.004

Press contact

Dr Louisa Wood, Communications Manager, Babraham Institute, louisa.wood@babraham.ac.uk

Image description

Repeat images of a two-cell stage mouse embryo (full image left, zoomed in image right) showing the expression of regulators of zygotic genome activation. The yellow areas are caused by overlap of Dppa2 (green) and Smarca5 (red) fluorescence. Image: Fátima Santos, Reik lab.

Affiliated authors (in author order):

Celia Alda-Catalinas, former PhD student, Reik lab

Irene Hernando-Herraez, postdoc researcher, Reik lab

Fátima Santos, senior research scientist, Reik lab

Oana Kubinyecz, PhD student, Reik lab

Mélanie Eckersley-Maslin, BBSRC Discovery fellow, Reik lab

Wolf Reik, group leader in the Epigenetics research programme

Research funding

Celia Alda-Catalinas was supported by a postgraduate award by the Biotechnology and Biological Sciences Research Council (BBSRC). Irene Hernando-Herraez is supported by a Marie Sklodowska-Curie Individual Fellowship. Melanie Eckersley-Maslin is supported by a BBSRC Discovery Fellowship. Research in the Reik lab is supported by BBSRC, the Wellcome Trust and the EU EpiGeneSys Network of Excellence.

Additional/related resources:

News, 22 June 2020 Early preparation allows genes to ‘come online’ later

10x Genomics blog post by Celia Alda-Catalinas, 16 March 2020: Resolving the transcriptional events of life’s earliest stages with CRISPR and single cell RNA-sequencing

News, 28 January 2019 Kick-starting the genome in early development

Animal research statement

As a publicly funded research institute, the Babraham Institute is committed to engagement and transparency in all aspects of its research. The research presented here is an example of how cell culture can replace the use of animals in research. The mouse embryonic stem cells used in this research were obtained from maintained stem cells lines and the use of the 2C-like cells (mimicking the mouse embryo from day 1.5 post fertilisation) allows investigation into a very early stage of development where it would be impractical to obtain the cells from live animals due to the numbers required for a screen.

Please follow the link for further details of our animal research and our animal welfare practices.