Publication Date
1-5-2022
Journal
Molecular Therapy
DOI
10.1016/j.ymthe.2021.10.023
PMID
34695545
PMCID
PMC8753568
PubMedCentral® Posted Date
10-23-2021
PubMedCentral® Full Text Version
Post-print
Published Open-Access
yes
Keywords
Animals, CRISPR-Cas Systems, Gene Editing, Lung, Mice, RNA, Guide, CRISPR-Cas Systems, Reproducibility of Results, lung editing, AAV5, CRISPR, Cas9, club cells, ciliated cells
Abstract
Genome editing in the lung has the potential to provide long-term expression of therapeutic protein to treat lung genetic diseases. Yet efficient delivery of CRISPR to the lung remains a challenge. The NIH Somatic Cell Genome Editing (SCGE) Consortium is developing safe and effective methods for genome editing in disease tissues. Methods developed by consortium members are independently validated by the SCGE small animal testing center to establish rigor and reproducibility. We have developed and validated a dual adeno-associated virus (AAV) CRISPR platform that supports effective editing of a lox-stop-lox-Tomato reporter in mouse lung airway. After intratracheal injection of the AAV serotype 5 (AAV5)-packaged S. pyogenes Cas9 (SpCas9) and single guide RNAs (sgRNAs), we observed ∼19%-26% Tomato-positive cells in both large and small airways, including club and ciliated epithelial cell types. This highly effective AAV delivery platform will facilitate the study of therapeutic genome editing in the lung and other tissue types.
Graphical Abstract
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Biochemistry, Biophysics, and Structural Biology Commons, Biology Commons, Medical Sciences Commons, Medical Specialties Commons
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