Simple Meets Single: The Application of CRISPR/Cas9 in Haploid Embryonic Stem Cells

Figure 2

The limitation of CRISPR/Cas9 in genetic screening. Each circle or oval represents a cell. 3n and 3n+1 and 3n+2 in yellow circles and orange and green ovals, respectively, represent the different types of InDels caused by a specific sgRNA. Grey circles are cells harboring other sgRNAs, but not the specific sgRNA mentioned above. In the first genetic screening (selection I), the disruption of the gene targeted by the specific sgRNA allows cell survival. Thus, only cells with 3n+1 and 3n+2 InDels, but not cells with 3n InDels, will survive after the selection. The integrated sgRNA sequence can be easily identified from the surviving cells. In the second genetic screening (selection II), the disruption of the gene targeted by the specific sgRNA renders cells to grow slower. To identify sgRNA causing the slow growth of the phenotype, high-throughput sequencing of the integrated sgRNA sequence and quantification of the relative amount of individual sgRNAs are required. However, cells with 3n InDels and an unchanged growth rate will interfere with the quantification, because cells with 3n, 3n+1, and 3n+2 InDels have the same sgRNA. Therefore, the observed reduction of cells with the specific sgRNA (including 3n, 3n+1, and 3n+2) is less than the actual reduction of cells with a disruption mutation (3n+1 and 3n+2). Therefore, the 3n mutation might lead to a false-negative hit.