High-throughput CRISPR Screening identifies key genes for LNP delivery

A flow cytometry-based genome-wide CRISPR knockout (KO) screen was developed to identify the pivotal genes that account for the variance in mRNA transfection efficiency observed between PL32 and P-UA LNPs. A549 cells were transduced with an H3 lentivirus library encompassing a broad spectrum of guide RNAs (gRNAs), targeting over 18,000 distinct genes with a total of 108,000 unique gRNAs. The cells were exposed to EGFP mRNA encapsulated within both PL32 and P-UA LNPs for 24 h. Subsequently, the cells were stratified into four groups based on their GFP expression levels, ranging from low to high. Genomic DNA was extracted from each of the sorted groups, and the sgRNA barcodes were amplified via PCR and sequenced.