br Introduction Retinoblastoma is the most

Introduction Retinoblastoma is the most common primary intraocular pediatric cancer, with 95% of the cases diagnosed before 5 years of age (Kivela, 2009). Retinoblastoma is initiated by biallelic inactivation of the retinoblastoma 1 (RB1) gene. In heritable retinoblastoma, a primary germline mutation in RB1 predisposes a child to retinal tumors, but only a second, somatic mutation in retinal cells initiates tumor growth (Goodrich, 2006). This heritable condition is responsible for all cases of bilateral retinoblastoma, i.e., involving tumors in both eyes (Dimaras et al., 2012; Richter et al., 2003). In up to 6% of patients, the inherited disease is manifested as trilateral retinoblastoma (TRb), where in addition to ocular tumors a primary neuroectodermal tumor also occurs (Dimaras et al., 2012). RB1 encodes for the retinoblastoma protein (pRB), a tumor suppressor and a key regulator of cell cycle (Manning and Dyson, 2012). pRB was initially shown to regulate cell-cycle progression through binding of the transcriptional activator E2F transcription factor 1 (E2F1). pRB phosphorylation by cyclin-dependent kinases prevents this association, allowing cell-cycle progression (Dynlacht et al., 1994; Weinberg, 1995). However, it is now known that pRB binds numerous cofactors, and is involved in many cellular mechanisms such as apoptosis, genome stability maintenance, and differentiation (Benavente and Dyer, 2015; Burkhart and Sage, 2008; Dyson, 2016; Thomas et al., 2003). Rb1-deficient mouse embryos exhibit substantial developmental defects in mesenchymal development, bone formation, hematopoiesis, and the nervous system (Calo et al., 2010; Jacks et al., 1992). Conditional knockout mice revealed pRb roles in neural migration and neurogenesis during development and adulthood (Andrusiak et al., 2011; Ghanem et al., 2012). In humans, deletions in chromosome 13q including RB1 were associated with nf-κb inhibitor abnormalities (Mitter et al., 2011; Rodjan et al., 2010), suggesting that it can play a role in human nervous system development. While an inherited heterozygous mutation in RB1 is the underlying cause of one-third of retinoblastoma cases, no cases of inherited homozygous inactivating mutations have been documented. Previous attempts to model retinoblastoma in mice were only partially successful, as Rb1 ablation in mice is embryonic lethal, and Rb1-null chimeras do not recapitulate basic features of human retinoblastoma (Jacks et al., 1992). Other studies used mutations in additional murine genes alongside Rb1 to initiate retinoblastoma or model the role of Rb1 in its initiation, diverging from its manifestation in humans (Classon and Harlow, 2002; Conklin et al., 2012). Human embryonic stem cells (hESCs) are normal primary cells with an indefinite self-renewal capability and the potential to differentiate toward any cellular fate. These properties make hESCs extremely beneficial for the study of developmental processes and disease modeling (Avior et al., 2016). In addition, hESCs share cellular characteristics with cancer cells (Ben-David and Benvenisty, 2011), suggesting that they may also be useful in modeling tumorigenic diseases. We therefore chose hESCs as a platform to model biallelic RB1 inactivation and TRb.
Results We used the CRISPR/Cas9 gene-editing approach to generate hESCs with mutations in RB1. Karyotypically normal hESCs were transfected either with the Cas9 gene alongside a guide RNA targeting the first exon of RB1, or with Cas9 only as control. The integrity of RB1 was then evaluated in individual clones using direct DNA sequencing, revealing two clones carrying a mutation in one allele (RB1+/−) and three clones with mutations in both alleles (RB1−/−) (Figures 1A and S1A). Western blot analysis validated the absence of pRB in the homozygous clones, whereas heterozygous clones maintained pRB expression as expected (Figures 1B and S1B). As pRB was previously shown to be important for chromosomal stability (Manning et al., 2010), genetic integrity of all clones was verified (Figure S1C).