RESUMEN
Double-strand breaks (DSBs) are the most toxic type of DNA lesions. Cells repair these lesions using either end protection- or end resection-coupled mechanisms. To study DSB repair choice, we present the Color Assay Tracing-Repair (CAT-R) to simultaneously quantify DSB repair via end protection and end resection pathways. CAT-R introduces DSBs using CRISPR/Cas9 in a tandem fluorescent reporter, whose repair distinguishes small insertions/deletions from large deletions. We demonstrate CAT-R applications in chemical and genetic screens. First, we evaluate 21 compounds currently in clinical trials which target the DNA damage response. Second, we examine how 417 factors involved in DNA damage response influence the choice between end protection and end resection. Finally, we show that impairing nucleotide excision repair favors error-free repair, providing an alternative way for improving CRISPR/Cas9-based knock-ins. CAT-R is a high-throughput, versatile assay to assess DSB repair choice, which facilitates comprehensive studies of DNA repair and drug efficiency testing.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de la Ataxia Telangiectasia Mutada/genética , Ciclo Celular , Supervivencia Celular , Daño del ADN , Reparación del ADN por Unión de Extremidades , Evaluación Preclínica de Medicamentos , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Poli(ADP-Ribosa) Polimerasa-1/genéticaRESUMEN
Arrayed CRISPR-based screens emerge as a powerful alternative to pooled screens making it possible to investigate a wide range of cellular phenotypes that are typically not amenable to pooled screens. Here, we describe a solid-phase transfection platform that enables CRISPR-based genetic screens in arrayed format with flexible readouts. We demonstrate efficient gene knockout upon delivery of guide RNAs and Cas9/guide RNA ribonucleoprotein complexes into untransformed and cancer cell lines. In addition, we provide evidence that our platform can be easily adapted to high-throughput screens and we use this approach to study oncogene addiction in tumor cells. Finally demonstrating that the human primary cells can also be edited using this method, we pave the way for rapid testing of potential targeted therapies.