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-nucleotidevariant clonal cell lines.

Functional EPAS1 / HIF2A missense variant is associated with hematocrit in Andean highlanders

“…15070063), at 37°C with 5% CO2. Transfection of plasmid DNA into HEK293T cells for the purpose of generating isogenic cell lines has been previously described ( 79 ). Briefly, cells were plated at a density of 50,000 cells per well in 250 μl of P/S-free medium per well in 48-well plates and transfected 16 hours after plating.…”

“…The 96-well plate was immediately placed into a tissue culture incubator after sorting. Subculturing and genotyping of single cells after clonal expansion was conducted as previously described ( 79 ). Genotyping of 26 clones confirmed the presence of three clones containing a heterozygous genotype and one clone containing a homozygous genotype (fig.…”

Successful Gene Editing of Apolipoprotein E4 to E3 in Brain of Alzheimer Model Mice After a Single IV Dose of Synthetic Exosome-Delivered CRISPR

“…40 CRISPR-CBE can be used to convert the 'C' in E4 to 'T' in E3, an irreversible DNA conversion, without requiring double-stranded DNA (dsDNA) breaks by using a Cas9 nickase (Cas9n). 41,42 Cas9n and cytidine deaminase enzyme fusion proteins, along with endogenous DNA repair systems, are used to effect the 'C'-'T' substitution, leading to correction of ~15-75% of total in vitro cellular DNA with minimal (≤ 1%) INDEL formation and minimal off-target editing vitro. 34,41 In addition, CBE allows editing in both dividing and non-dividing cells such as brain neurons.…”

Curing “GFP-itis” in Bacteria with Base Editors: Development of a Genome Editing Science Program Implemented with High School Biology Students

“…However, pre-college students are less aware of newer genome engineering tools like base editors (BEs). BEs enable scientists to introduce point mutations at targeted sites in the genome of living cells with high efficiency and precision (21,22) and thus have the therapeutic potential to treat thousands of human genetic disorders (Figure 1A-B) (23)(24)(25). A hands-on laboratorybased experiment that uses base editing would introduce students to cutting-edge genome editing technologies while building upon previous knowledge of CRISPR technologies (as BEs are modified CRISPR systems).…”