citations

                    Highly scalable arrayed CRISPR mediated gene silencing in primary lungsmall airway epithelial cells
                

Authors:

Dickson A, Mullooly N, Serrano A, Escudero-Ibarz L, Wiggins C, Gianni D

In:

Source: SLAS Discov
Publication Date: (2023)
Issue: 2: 29-35

Research Area:

Basic Research

Respiratory Research

Drug Discovery

Cells used in publication:

Epithelial, Small Airway, human (SAEC): Species: human; Tissue Origin: lung

Culture Media:

Small Airway Epithelial Cell Growth Medium

Platform:

384-well HT Nucleofector® System

Experiment

Electroporation a. Bravo transfer 20 µl of cells/RNP to a new 384 well electroporation cassette. The following transfer steps are recommended > Use 70 µl tips > Mix 10 µl (1 x mix) in RNP plate at a slow setting > Aspirate 21 µl from RNP plate > Dispense 20 µl to electroporation cassette. b. Transfer electroporation plate to the 384 HT nucleofector system. Adjust the settings for a 20 µl electroporation reaction and use programme CM-138-AA. A successful electroporation event is indicated by green wells on the software. c. The electroporated cells are now ready for Bravo transfer to assay plates. CRITICAL: It is critical that there are no bubbles in the wells of the electroporation plate. This may result in a failed electroporation well which will be indicated by an orange/red well on the software ( see troubleshooting problem 3 ). Bubbles may be caused by presence of serum in the P3 electroporation cocktail, so a PBS wash step is included to prevent serum transfer. Bubbles may additionally be caused by the Bravo transfer. It is recommended in the protocol to aspirate 21 µl from the RNP plate and dispense 20 µl to the cassette to avoid any air bubbles. It is critical that cells are not centrifuged in P3 buffer. Optional: The protocol has been designed for an arrayed CRISPRn screen in a full 384 well plate. Volumes can be adjusted if more or less wells are required. Note : The automation steps, including using the Thermo Scientific Multidrop Combi to dispense cells to the RNP plate and automated transfer, have been optimised to increase robustness and reproducibility of the protocol. It is possible to do these steps manually with a multi- channel pipette if required. Note : Prior to editing, the viability of the SAECs should be assessed whilst carrying out cell counting. For the editing to be successful the viability of the SAECs should be > 70%. Pause point 3 : Following detachment and pooling of cell pellets to a final volume of 20 mL in media, the protocol can be paused for approx. 30 minutes while the cells remain in fresh media.

Abstract

Small airway epithelial cells (SAECs) play a central role in the pathogenesis of lung diseases and are now becoming a crucial cellular model for target identification and validation in drug discovery. However, primary cell lines such as SAECs are often difficult to transfect using traditional lipofection methods; therefore, gene editing using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is often carried out through ribonucleoprotein (RNP) electroporation. Here we have established a robust, scalable, and automated arrayed CRISPR nuclease (CRISPRn) screening workflow for SAECs which can be combined with a myriad of disease-specific endpoint assays.

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