Red Alert

Mechanistic insight into alveolar type 2 (AT2) cell dysfunction has come largely from murine genetics, induced pluripotent stem cell–derived AT2 models, and viral transduction of primary human AT2 cells. While valuable, current approaches for genetic modification of human AT2 cells models are slow and expensive. Tristan Kooistra, MD, Tresa Sarraf, PhD (Instructor and Postdoctoral Researcher, respectively, Massachusetts General Hospital), and colleagues describe a nonviral, selection-free alternative: electroporation of preassembled CRISPR-Cas ribonucleoprotein (RNP) complexes into dissociated AT2 organoids. Screening 18 electroporation programs for protein-level β2-microglobulin deletion identified conditions achieving 75% to 85% knockout with Cas9 while preserving yield, colony-forming efficiency, organoid size, and expression of the key AT2 markers SPC and HTII-280. Cas12a RNPs improved efficiency to near-complete knockout across 3 donors, holding above 95% even at >10-fold RNP dilution, with comparable results when targeting CD63. Knockout phenotypes can now be read out in 1 to 2 weeks using commercial reagents, at an order of magnitude lower cost than viral methods. This method should also extend to multiplexed editing, deletion of noncoding regulatory elements, homology-directed repair–based knock-in, and clonal screening, making the human alveolar epithelium more accessible to genetic dissection.