Key result
CRISPRi Perturb-seq protocol identifies critical genes driving in vitro cardiomyocyte differentiation.
Why the study?
Large-scale Perturb-Seq experiments combining CRISPR screening with scRNA-seq are costly, and applying them during development is complicated by differentiation heterogeneity.
This protocol enables large-scale, cost-effective identification of genes required for cardiomyocyte development using CRISPR-based screening combined with single-cell RNA sequencing.
While Perturb-Seq combines CRISPR-based screening with single-cell RNA sequencing (scRNA-seq), large-scale experiments are costly and its application during development is complicated by differentiation heterogeneity. Here, we present a protocol to identify genes required for cardiomyocyte development using Perturb-Seq. We describe steps for sgRNA (single guide RNA) library cloning and infection, cardiomyocyte differentiation, cell hashing, super loading, and scRNA-seq. We then detail procedures for sequencing, mapping, and data analysis. For complete details on the use and execution of this protocol, please refer to Sivakumar et al. 1
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Sivakumar et al. (2026) studied Cardiomyocyte development. Perturb-Seq (CRISPRi followed by scRNA-seq) was evaluated. A detailed protocol was developed for performing CRISPR interference Perturb-Seq to identify genes required for cardiomyocyte development during in vitro differentiation from pluripotent stem cells.
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