Alexa E. Martinez

Bio: Alexa E. Martinez is an academic researcher from Baylor College of Medicine. The author has contributed to research in topics: Cas9 & Genome. The author has co-authored 1 publications.

Progress of delivery methods for CRISPR-Cas9

Abstract: ABSTRACT Introduction Gene therapy is becoming increasingly common in clinical practice, giving hope for the correction of a wide range of human diseases and defects. The CRISPR/Cas9 system, consisting of the Cas9 nuclease and single-guide RNA (sgRNA), has revolutionized the field of gene editing. However, efficiently delivering the CRISPR-Cas9 to the target organ or cell remains a significant challenge. In recent years, with rapid advances in nanoscience, materials science, and medicine, researchers have developed various technologies that can deliver CRISPR-Cas9 in different forms for in vitro and in vivo gene editing. Here, we review the development of the CRISPR-Cas9 and describe the delivery forms and the vectors that have emerged in CRISPR-Cas9 delivery, summarizing the key barriers and the promising strategies that vectors currently face in delivering the CRISPR-Cas9. Areas covered With the rapid development of CRISPR-Cas9, delivery methods are becoming increasingly important in the in vivo delivery of CRISPR-Cas9. Expert opinion CRISPR-Cas9 is becoming increasingly common in clinical trials. However, the complex nuclease and protease environment is a tremendous challenge for in vivo clinical applications. Therefore, the development of delivery methods is highly likely to take the application of CRISPR-Cas9 technology to another level.

rAAV-MEDIATED EDITING OF THE G551D CFTR MUTATION IN FERRET AIRWAY BASAL CELLS.

Abstract: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), a chronic disease that affects multiple organs including the lung. We developed a CF ferret model of a scarless G551→D substitution in CFTR (CFTRG551D-KI), enabling approaches to correct this gating mutation in CF airways via gene editing. Homology-directed repair (HDR) was tested in Cas9-expressing CF airway basal cells (Cas9-GKI) from this model, as well as reporter basal cells (Y66S-Cas9-GKI) that express an integrated non-fluorescent Y66S-EGFP mutant gene to facilitate rapid assessment of HDR by the restoration of fluorescence. rAAV vectors were used to deliver two DNA templates and sgRNAs for dual gene editing at the EGFP and CFTR genes, followed by fluorescence activated cell sorting (FACS) of EGFPY66S-corrected cells. When gene-edited airway basal cells were polarized at an air-liquid interface, unsorted and EGFPY66S-corrected sorted populations gave rise to 26.0% and 70.4% CFTR-mediated Cl- transport of that observed in non-CF cultures, respectively. The consequences of gene editing at the CFTRG551D locus by HDR and non-homology end joining (NHEJ) were assessed by targeted gene next generation sequencing (NGS) against a specific amplicon. NGS revealed HDR corrections of 3.1% of G551 sequences in the unsorted population of rAAV-infected cells, and 18.4% in the EGFPY66S-corrected cells. However, the largest proportion of sequences had indels surrounding the CRISPR cut site, demonstrating that NHEJ was the dominant repair pathway. This approach to simultaneously co-edit at two genomic loci using rAAV may have utility as a model system for optimizing gene editing efficiencies in proliferating airway basal cells through the modulation of DNA repair pathways in favor of HDR.