CRISPR-Cas9 Gene Editing to Generate Isoform-Specific LAMP-2A Knockout in Human Cancer Cells.

Chaperone-mediated autophagy (CMA) is a highly specific lysosomal-dependent protein degradation pathway. A critical molecular component of CMA is the lysosome-associated membrane protein (LAMP) type 2A, which is required for substrate uptake by the lysosome. Defects in the CMA pathway have been associated with various human pathologies, including malignancies, increasing the overall interest in methods to monitor this selective autophagy process. Yet isogenic LAMP-2A knockout cancer cell models are still lacking. This is likely to depend on challenges related to that human LAMP-2 gene undergoes alternative splicing of its pre-mRNA, generating three isoform variants, LAMP-2A, LAMP-2B, and LAMP-2C. However, without assessment of the impact of LAMP-2A loss of function specifically in human cells, the involvement of CMA in human pathologies, including carcinogenesis remains speculative. Here, we describe the generation of isoform-specific CRISPR-Cas9 genomic editing of LAMP-2A in human cancer cells, without affecting the other two isoforms, allowing for experimental evaluation of LAMP-2A, thus CMA in human cancer models.