Validation of gene editing efficiency with CRISPR-Cas9 system directly in rat zygotes using electroporation mediated delivery and embryo culture

Successful use of the CRISPR-Cas9 system for gene manipulation relies on identifying effective and efficient guide RNA sequences (gRNAs). When the goal is to create transgenic animal/rodent models by knocking-in desired sequences using homology-directed repair (HDR), selecting effective guides becomes even more critical to minimize developmental time and resources. Currently, validation experiments for gRNAs for generating rat models are carried out using immortalized rat cells. However, there are several limitations with using such cell lines, including ploidy of the genome, non-predictive transfection efficiency, and the ability to identify gene modifications efficiently within diverse cell populations. Since embryos are authentic representatives of live animals compared to cell lines, validating CRISPR guides for their nuclease activity in freshly isolated embryos will provide greater accuracy of in vivo gene editing efficiency. In contrast to microinjections, delivery by electroporation is a more accessible method that can be simple and does not require special skills and equipment. We demonstrate an accessible workflow to either delete or edit target genes in vivo in rats using the efficient editing of a human mutation in alpha7 nicotinic acetylcholine receptor subunit (CHRNA7) ortholog using electroporation as a delivery method for CRISPR-Cas9 ribonucleoprotein complexes in rat embryos. • Upon identifying CRISPR targets at the desired genetic alteration site, we designed homologydriven repair (HDR) templates for effective and easy identification of gene editing by Restriction Fragment Length Polymorphism (RFLP). • Cultured rat embryos can be genotyped to assess CRISPR activity as seen by either presence of indels resulting from NHEJ or knock-in of repair template resulting from homology driven repair. • Heteroduplex mobility assay (HMA) and Restriction Fragment Length Polymorphism (RFLP) of PCR products can be performed reliably and reproducibly at a low-cost

Use of ferrets for electrophysiologic monitoring of ion transport

<div><p>Limited success achieved in translating basic science discoveries into clinical applications for chronic airway diseases is attributed to differences in respiratory anatomy and physiology, poor approximation of pathologic processes, and lack of correlative clinical endpoints between humans and laboratory animal models. Here, we discuss advantages of using ferrets (<i>Mustela putorus furo)</i> as a model for improved understanding of human airway physiology and demonstrate assays for quantifying airway epithelial ion transport <i>in vivo</i> and <i>ex vivo</i>, and establish air-liquid interface cultures of ferret airway epithelial cells as a complementary <i>in vitro</i> model for mechanistic studies. We present data here that establishes the feasibility of measuring these human disease endpoints in ferrets. Briefly, potential difference across the nasal and the lower airway epithelium in ferrets could be consistently assessed, were highly reproducible, and responsive to experimental interventions. Additionally, ferret airway epithelial cells were amenable to primary cell culture methods for <i>in vitro</i> experiments as was the use of ferret tracheal explants as an <i>ex vivo</i> system for assessing ion transport. The feasibility of conducting multiple assessments of disease outcomes supports the adoption of ferrets as a highly relevant model for research in obstructive airway diseases.</p></div

Measurement of short-circuit current (Isc) in cultured ferret bronchial epithelial cells and in explants of ferret trachea.

<p><b>A</b>: Representative hematoxylin and eosin stained image of a well-differentiated ferret bronchial epithelial cells grown at air-liquid interface. <b>B</b>: Representative Isc tracings of primary ferret bronchial epithelial cells sequentially exposed to forskolin (10 μM), followed by GlyH101 (20 μM) in the setting of amiloride (100 μM) and a chloride secretory gradient. <b>C</b>: Summary data of Isc measurements from different ALI cultures indicating stimulated Isc following acute addition of forskolin or GlyH101. N  =  4/condition. <b>D</b>: Representative Isc tracing of an <i>ex vivo</i> ferret trachea sequentially exposed to Ringer’s, chloride-free, forskolin (20 μM), followed by GlyH101 (20 μM) in the setting of amiloride (100 μM). <b>E</b>: Summary data of Isc measurements from different trachea showing stimulated Isc following acute addition of amiloride, forskolin, or GlyH101. N = 7/condition.</p

Potential difference measurements across nasal epithelium.

<p>Potential difference measurements across nasal epithelium.</p

Potential difference measurements across lower airway epithelium.

<p>Potential difference measurements across lower airway epithelium.</p

Schematic of laboratory set up for potential difference (PD) measurements in ferrets.

<p>Cartoon representing hardware and electrical connections representing PD apparatus modified from the human instrument recommended by the standard operating procedure of the CF Therapeutics Development Network and the European Clinical Trial Network [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186984#pone.0186984.ref019" target="_blank">19</a>]. Ground Lead: Connects electrical apparatus to ground for reference from which voltage is measured. Headstage: Connects electrode inputs to bioamplifier and safely isolates from wall AC current. Bioamplifier: Serves as voltmeter to measure PD. Analog-Digital Converter: Converts analog signals to digital form. K/Cl Calomel Electrodes: Measure PD. Agar Bridge: Connects positively charged (+) calomel electrodes to ferret nasal epithelium or lower airway epithelium through Airway Catheter and negatively charged (-) calomel electrodes to subcutaneous tissue through Butterfly Needle. Syringe Pumps: Deliver test solutions perfused sequentially from pump 1 to 5 with a flow rate of 4ml/hr (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186984#pone.0186984.s002" target="_blank">S1 Table</a>). The PD changes are observed and recorded on the computer using LabChart 7.0 software. Following the procedure, the nasal cannula and subcutaneous needle were removed, and anesthesia reversed with atipamezole (5 mg/kg body weight, IM) delivered at an equal volume as dexmedetomidine. All ferrets recovered within 10–15 min, and remained on a warming pad until aroused and moving. Supplemental oxygen was not required during the NPD procedures in our experience, however supplemental oxygen was delivered during recovery from anesthesia.</p

Measurement of lower airway potential difference (LAPD) in wild type ferrets.

<p><b>A</b>. Representative LAPD tracing in a sedated intubated ferret indicating PD changes following perfusion with Ringer’s solution, amiloride (100 μM), chloride-free, chloride-free + forskolin (20 μM), and CFTR-specific inhibitor GlyH101 (10 μM); N = 5. <b>B</b>: Summary tracing of PD changes in ferrets following infusion of the 5 sequential reagents, N = 5. <b>C</b>: Change in LAPD measurements from 5 (male and female) adult, wild type ferrets.</p