Colorectal cancer (CRC) is a major health burden in Australia, causing more than 4,000 deaths every year. Current first-line chemotherapy treatment involves a combination of chemotherapeutic agents that includes 5-fluorouracil (5-FU), which kills actively cycling tumour cells. However, for patients that initially respond to treatment, the benefits are usually short lived as the tumours relapse and develop chemoresistance. Therefore, defining the molecular mechanisms underlying the emergence of resistance for effective treatment of CRC is urgently needed. We recently discovered that expression of high levels of clusterin (CLU) is associated with both lower patient survival and higher disease recurrence in CRC. CLU has been identified as a marker for a quiescent intestinal stem cell population which is responsible for repairing the injured intestine. Therefore, we hypothesize that CLU-expressing stem cells exhibit plasticity to promote chemoresistance in CRC. We have made use of our extensive collection of CRC tissues and patient-derived colorectal cancer organoids to characterise the CLU-expressing cells in CRC. Immunohistochemistry on CRC tissue sections and patient-derived colorectal cancer organoids confirmed the existence of CLU-expressing cells in patient’s CRC tissues. We also discovered that CLU is significantly enriched following 5-FU treatment of patient-derived colorectal cancer organoids and the expression strongly correlates with the level of drug resistance. We then further investigate the role of CLU-expressing cells in chemoresistance by utilizing the latest CRISPR–HOT technology to robustly and rapidly genetically modify CLU-expressing cells in CRC organoids to incorporate fluorescent tags, to permit visualisation, isolation, lineage tracing and specific ablation of CLU-expressing cells. This study will provide insight into the role of CLU-expressing cells in driving resistance to common treatments for CRC and highlight the potential of using CLU-expressing cells as a predictive marker of drug resistance as well as a potential therapeutic target.