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Title: Base Editing in Human Cells to Produce Single‐Nucleotide‐Variant Clonal Cell Lines
Abstract

Base‐editing technologies enable the introduction of point mutations at targeted genomic sites in mammalian cells, with higher efficiency and precision than traditional genome‐editing methods that use DNA double‐strand breaks, such as zinc finger nucleases (ZFNs), transcription‐activator‐like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR‐associated protein 9 (CRISPR‐Cas9) system. This allows the generation of single‐nucleotide‐variant isogenic cell lines (i.e., cell lines whose genomic sequences differ from each other only at a single, edited nucleotide) in a more time‐ and resource‐effective manner. These single‐nucleotide‐variant clonal cell lines represent a powerful tool with which to assess the functional role of genetic variants in a native cellular context. Base editing can therefore facilitate genotype‐to‐phenotype studies in a controlled laboratory setting, with applications in both basic research and clinical applications. Here, we provide optimized protocols (including experimental design, methods, and analyses) to design base‐editing constructs, transfect adherent cells, quantify base‐editing efficiencies in bulk, and generate single‐nucleotide‐variant clonal cell lines. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Design and production of plasmids for base‐editing experiments

Basic Protocol 2: Transfection of adherent cells and harvesting of genomic DNA

Basic Protocol 3: Genotyping of harvested cells using Sanger sequencing

Alternate Protocol 1: Next‐generation sequencing to quantify base editing

Basic Protocol 4: Single‐cell isolation of base‐edited cells using FACS

Alternate Protocol 2: Single‐cell isolation of base‐edited cells using dilution plating

Basic Protocol 5: Clonal expansion to generate isogenic cell lines and genotyping of clones

 
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NSF-PAR ID:
10238444
Author(s) / Creator(s):
 ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Current Protocols in Molecular Biology
Volume:
133
Issue:
1
ISSN:
1934-3639
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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