Impact of gene editing on the study of cystic fibrosis

Harrison, Patrick T.; Sanz, David J.; Hollywood, Jennifer A.

doi:10.1007/s00439-016-1693-3

Cited by 17 publications

(18 citation statements)

References 103 publications

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“…More than 120 different mutations of the CFTR gene are known, definitely causing milder or more severe disease (2). The most abundant mutation is the ΔF508 mutation (lack of phenylalanine at position 508) (2,3).…”

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confidence: 99%

Bactericidal and Fungicidal Activity of N -Chlorotaurine Is Enhanced in Cystic Fibrosis Sputum Medium

Gruber

Moser

Nagl

et al. 2017

Antimicrob Agents Chemother

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Lung infections with multiresistant pathogens are a major problem among patients suffering from cystic fibrosis (CF). N-Chlorotaurine (NCT), a microbicidal active chlorine compound with no development of resistance, is well tolerated upon inhalation. The aim of this study was to investigate the in vitro bactericidal and fungicidal activity of NCT in artificial sputum medium (ASM), which mimics the composition of CF mucus. The medium was inoculated with bacteria (Staphylococcus aureus, including some methicillin-resistant S. aureus [MRSA] strains, Pseudomonas aeruginosa, and Escherichia coli) or spores of fungi (Aspergillus fumigatus, Aspergillus terreus, Candida albicans, Scedosporium apiospermum, Scedosporium boydii, Lomentospora prolificans, Scedosporium aurantiacum, Scedosporium minutisporum, Exophiala dermatitidis, and Geotrichum sp.), to final concentrations of 10 7 to 10 8 CFU/ml. NCT was added at 37°C, and time-kill assays were performed. At a concentration of 1% (10 mg/ml, 55 mM) NCT, bacteria and spores were killed within 10 min and 15 min, respectively, to the detection limit of 10 2 CFU/ml (reduction of 5 to 6 log 10 units). Reductions of 2 log 10 units were still achieved with 0.1% (bacteria) and 0.3% (fungi) NCT, largely within 10 to 30 min. Measurements by means of iodometric titration showed oxidizing activity for 1, 30, 60, and Ͼ60 min at concentrations of 0.1%, 0.3%, 0.5%, and 1.0% NCT, respectively, which matches the killing test results. NCT demonstrated broad-spectrum microbicidal activity in the milieu of CF mucus at concentrations ideal for clinical use. The microbicidal activity of NCT in ASM was even stronger than that in buffer solution; this was particularly pronounced for fungi. This finding can be explained largely by the formation, through transhalogenation, of monochloramine, which rapidly penetrates pathogens.

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confidence: 99%

Bactericidal and Fungicidal Activity of N -Chlorotaurine Is Enhanced in Cystic Fibrosis Sputum Medium

Gruber

Moser

Nagl

et al. 2017

Antimicrob Agents Chemother

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“…However, since these enrichment strategies are not compatible with direct in vivo application of gene editing16, we decided to further characterise template-dependent editing without using a selection approach. Here, we describe a CRISPR Cas9/gRNA strategy to allow the correction of the F508del mutation and characterisation of repair tracts either side of the Cas9-induced DSB by deep sequencing.…”

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confidence: 99%

Analysis of gene repair tracts from Cas9/gRNA double-stranded breaks in the human CFTR gene

Hollywood

Lee

Scallan

et al. 2016

Sci Rep

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To maximise the efficiency of template-dependent gene editing, most studies describe programmable and/or RNA-guided endonucleases that make a double-stranded break at, or close to, the target sequence to be modified. The rationale for this design strategy is that most gene repair tracts will be very short. Here, we describe a CRISPR Cas9/gRNA selection-free strategy which uses deep sequencing to characterise repair tracts from a donor plasmid containing seven nucleotide differences across a 216 bp target region in the human CFTR gene. We found that 90% of the template-dependent repair tracts were >100 bp in length with equal numbers of uni-directional and bi-directional repair tracts. The occurrence of long repair tracts suggests that a single gRNA could be used with variants of the same template to create or correct specific mutations within a 200 bp range, the size of ~80% of human exons. The selection-free strategy used here also allowed detection of non-homologous end joining events in many of the homology-directed repair tracts. This indicates a need to modify the donor, possibly by silent changes in the PAM sequence, to prevent creation of a second double-stranded break in an allele that has already been correctly edited by homology-directed repair.

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“…Although several gene/ mRNA based strategies are currently being explored and developed, they are outside the scope of this article, which focusses only on CFTR modulating small molecules; the interested reader is directed towards a recent reviews (5,6). Three main groups of small molecules are in clinical development: potentiators, correctors and read-through compounds.…”

Section: Cftr Modulatorsmentioning

confidence: 99%