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.

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