Forward genetic screens in haploid mammalian stem cells: looking beyond CRISPR/Cas9

Recent adaptation of the CRISPR/Cas9 bacterial system to facilitate manipulation of mammalian genomes has provided a real breakthrough for genome editing applications. Development of whole-genome CRISPR libraries with the aim of generating gene knockouts for every single coding sequence has allowed forward genetic screening in mammalian cells with unprecedented efficiency and versatility. CRISPR/Cas9 approaches, however, rely on phenotypes associated with loss-of-function mutations. Single-nucleotide variations (SNVs), on the other hand, have the potential to uncover not only loss-of-function phenotypes (by generating nonsense mutations, for example) but also gain-of-function phenotypes through missense mutations. In addition, they produce valuable information regarding functionally important domains of the affected gene product, as SNVs causative of a particular phenotype tend to cluster around specific regions of the amino acid sequence of the encoded protein. SNV-based approaches in mammalian cells, however, have been hindered by the diploid nature of their genomes, a fact that complicates the establishment of straightforward genotype-to-phenotype correlations. In this talk I will discuss how we apply CRISPR/Cas9 genetic screening to further understand the DNA-damage response in mouse embryonic stem cells (mESCs), and I will also introduce the use of haploid mESCs to perform SNV-based forward genetic screens.