RESUMEN

Here we developed a complementation method for the study of essential genes in live human cells using the CRISPR/Cas9 system. Proteins encoded by essential genes were expressed using a derivative of the pCEP4 compensating plasmid in combination with Cas9 endonuclease targeting of the chromosomal genes. We show that this strategy can be applied to essential genes, such as those coding for proliferating cell nuclear antigen (PCNA) and DNA polymerase delta subunit 2 (POLD2). As demonstrated for the PCNA protein, our method allows mutational analysis of essential protein-coding sequences in live cells.

Asunto(s)

Sistemas CRISPR-Cas/genética , Análisis Mutacional de ADN/métodos , Genes Esenciales/genética , Prueba de Complementación Genética/métodos , Técnicas Citológicas , Técnicas de Inactivación de Genes , Humanos , Modelos Moleculares , Mutación/genética , Antígeno Nuclear de Célula en Proliferación/genética