Speaker
Description
Pooled CRISPR screenings are powerful functional genomics tools to assess how perturbing gene expression alters cellular fitness, differentiation, and transcriptomic landscapes in complex biological models. When studying systems that recapitulate brain development, it is crucial to resolve changes in developmental trajectories and cellular dynamics at single-cell resolution, as diverse neural cell types may respond differently to genetic perturbations. While tools like CROP-seq and Perturb-seq have advanced our understanding of gene effects on complex neurobiological traits, they primarily focus on single-gene perturbations. However, multiplexed CRISPR screening is essential for studying combinatorial interactions, yet current methodologies largely remain incompatible with pooled approaches requiring mRNA-embedded barcodes, thus limiting the possibility of pairing multiple genetic perturbations with morphological and transcriptome shifts at single-cell resolution.
Here, we present CROPseq-multi, a new construct enabling multiplexed Cas9-based perturbations while preserving mRNA barcoding of sgRNAs, allowing scRNA-seq and spatial in situ sequencing of perturbed cells. To validate this approach, we generated a lentiviral library delivering two sgRNAs per cell targeting 42 genes involved in neural progenitor differentiation. Additionally, we performed single and double knockout of GSK3A/B to investigate both individual and combinatorial perturbations of this key regulator of neural progenitor proliferation. Clonal barcodes were incorporated into the sgRNA sequence to track lineage dynamics throughout differentiation. We then transduced the library into stem cells and induced Cas9 expression during differentiation, challenging neural patterning with pooled gene knockouts.
In a two-phase pilot study, we benchmark the compatibility of CROPseq-multi with different sequencing strategies using both 3’ short- and long-read methods and 5’ targeted capture, ensuring compatibility with commercially available platforms without requiring modifications to sgRNA scaffolds.
This versatile solution provides the field with a new all-in-one vector that enhances functional genomics in neuroscience and beyond, unlocking new avenues for G × E studies and disease modelling at single-cell resolution.
| Author(s) | Manuel Lessi*, Russell Walton*, Nicolo Caporale, Paul Blainey, Giuseppe Testa |
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| Affiliation(s) | "Human Technopole, Viale Rita Levi-Montalcini 1, 20157, Milan, Italy Department of Oncology and Hemato-Oncology, University of Milan, Via Santa Sofia 9, 20122, Milan, Italy, Broad Institute of MIT and Harvard, Cambridge, MA, USA Department of Biological Engineering, MIT, Cambridge, MA, USA, Human Technopole, Viale Rita Levi-Montalcini 1, 20157, Milan, Italy Department of Oncology and Hemato-Oncology, University of Milan, Via Santa Sofia 9, 20122, Milan, Italy, Broad Institute of MIT and Harvard, Cambridge, MA, USA Department of Biological Engineering, MIT, Cambridge, MA, USA Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA, Human Technopole, Viale Rita Levi-Montalcini 1, 20157, Milan, Italy Department of Oncology and Hemato-Oncology, University of Milan, Via Santa Sofia 9, 20122, Milan, Italy Department of Experimental Oncology, European Institute of Oncology IRCCS, Via Adamello 16, 20139, Milan, Italy" |
