We present scMAGeCK, a computational framework to identify genomic elements associated with multiple expression-based phenotypes in CRISPR/Cas9 functional screening that uses single-cell RNA-seq as readout. scMAGeCK outperforms existing methods, identifies genes and enhancers with known and novel functions in cell proliferation, and enables an unbiased construction of genotype-phenotype network. Single-cell CRISPR screening on mouse embryonic stem cells identifies key genes associated with different pluripotency states. Applying scMAGeCK on multiple datasets, we identify key factors that improve the power of single-cell CRISPR screening. Collectively, scMAGeCK is a novel tool to study genotype-phenotype relationships at a single-cell level.

Most of the mammalian follicles undergo a degenerative process called “follicle atresia”. Apoptosis of granulosa cells is the main characteristic of follicle atresia. Follicle stimulating hormone (FSH) and the transforming growth factor β (TGF-β) superfamily have important regulatory functions in this process. FSH activates protein kinase A and cooperating with insulin receptor substrates, it promotes the PI3K/Akt pathway which weakens apoptosis. Both Smad or non-Smad signaling of the transforming growth factor β superfamily seem to be related to follicle atresia, and the effect of several important family members on follicle atresia is concluded in this article. FSH and TGF-β are likely to mutually influence each other and what we have already known about the possible underlying molecular mechanism is also discussed below.

C-terminus kinesin motor KIFC1 is known for centrosome clustering in cancer cells with supernumerary centrosomes. KIFC1 crosslinks and glides on microtubules (MT) to assist normal bipolar spindle formation to avoid multi-polar cell division, which might be fatal. Testis cancer is the most common human cancer among young men. However, the gene expression profiles of testis cancer is still not complete and the expression of the C-terminus kinesin motor KIFC1 in testis cancer has not yet been examined. We found that KIFC1 is enriched in seminoma tissues in both mRNA level and protein level, and is specifically enriched in the cells that divide actively. Cell experiments showed that KIFC1 may be essential in cell division, but not essential in metastasis. Based on subcellular immuno-florescent staining results, we also described the localization of KIFC1 during cell cycle. By expressing ΔC-FLAG peptide in the cells, we found that the tail domain of KIFC1 might be essential for the dynamic disassociation of KIFC1, and the motor domain of KIFC1 might be essential for the degradation of KIFC1. Our work provides a new perspective for seminoma research.

CRISPR/Cas9 based functional screening coupled with single-cell RNA-seq ("single-cell CRISPR screening") unravels gene regulatory networks and enhancer-gene regulations in a large scale. We propose scMAGeCK, a computational framework to systematically identify genes and non-coding elements associated with multiple expression-based phenotypes in single-cell CRISPR screening. scMAGeCK identified genes and enhancers that modulate the expression of a known proliferation marker, MKI67 (Ki-67), a result that resembles the outcome of proliferation-linked CRISPR screening. We further performed single-cell CRISPR screening on mouse embryonic stem cells (mESC), and identified key genes associated with different pluripotency states. scMAGeCK enabled an unbiased construction of genotypephenotype network, where multiple phenotypes can be regulated by different gene perturbations. Finally, we studied key factors that improve the statistical power of single-cell CRISPR screens, including target gene expression and the number of guide RNAs (gRNAs) per cell. Collectively, scMAGeCK is a novel and effective computational tool to study genotypephenotype relationships at a single-cell level.