Generating stably-expressing Cas9 cancer organoid lines v1

This protocol aims to establish a robust Cas9 expression system in cancer organoids through a three-stage process. The first stage involves titrating blasticidin to determine the optimal concentration for eliminating wild-type cells while supporting the survival and growth of Cas9-expressing cells. The second step introduces the Cas9 gene, and the specified blasticidin concentration selects and maintains Cas9-positive cells. The final stage assesses Cas9 activity, ensuring functionality is above 75%. This established system facilitates precise and genome-wide gene modification using the 'lentiCas9-Blast' CRISPR-Cas9 guide RNA system. Process Diagram