Development of multiplexed orthogonal base editor (MOBE) systems.

Base editors (BEs) enable efficient, programmable installation of point mutations while avoiding the use of double-strand breaks. Simultaneous application of two or more different BEs, such as an adenine BE (which converts A·T base pairs to G·C) and a cytosine BE (which converts C·G base pairs to T·A), is not feasible because guide RNA crosstalk results in non-orthogonal editing, with all BEs modifying all target loci. Here we engineer both adenine BEs and cytosine BEs that can be orthogonally multiplexed by using RNA aptamer-coat protein systems to recruit the DNA-modifying enzymes directly to the guide RNAs. We generate four multiplexed orthogonal BE systems that enable rates of precise co-occurring edits of up to 7.1% in the same DNA strand without enrichment or selection strategies. The addition of a fluorescent enrichment strategy increases co-occurring edit rates up to 24.8% in human cells. These systems are compatible with expanded protospacer adjacent motif and high-fidelity Cas9 variants, function well in multiple cell types, have equivalent or reduced off-target propensities compared with their parental systems and can model disease-relevant point mutation combinations.

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

Hypoxia-inducible factor pathway genes are linked to adaptation in both human and nonhuman highland species. EPAS1, a notable target of hypoxia adaptation, is associated with relatively lower hemoglobin concentration in Tibetans. We provide evidence for an association between an adaptive EPAS1 variant (rs570553380) and the same phenotype of relatively low hematocrit in Andean highlanders. This Andean-specific missense variant is present at a modest frequency in Andeans and absent in other human populations and vertebrate species except the coelacanth. CRISPR-base-edited human cells with this variant exhibit shifts in hypoxia-regulated gene expression, while metabolomic analyses reveal both genotype and phenotype associations and validation in a lowland population. Although this genocopy of relatively lower hematocrit in Andean highlanders parallels well-replicated findings in Tibetans, it likely involves distinct pathway responses based on a protein-coding versus noncoding variants, respectively. These findings illuminate how unique variants at EPAS1 contribute to the same phenotype in Tibetans and a subset of Andean highlanders despite distinct evolutionary trajectories.

Base Editors: Expanding the Types of DNA Damage Products Harnessed for Genome Editing.

Base editors are an innovative addition to the genome editing toolbox that introduced a new genome editing strategy to the field. Instead of using double-stranded DNA breaks, base editors use nucleobase modification chemistry to efficiently and precisely incorporate single nucleotide variants (SNVs) into the genome of living cells. Two classes of DNA base editors currently exist: deoxycytidine deamination-derived editors (CBEs, which facilitate C•G to T•A mutations) and deoxyadenosine deamination-derived base editors (ABEs, which facilitate A•T to G•C mutations). More recently, the development of mitochondrial base editors allowed the introduction of C•G to T•A mutations into mitochondrial DNA as well. Base editors show great potential as therapeutic agents and research tools, and extensive studies have been carried out to improve upon the original base editor constructs to aid researchers in a variety of disciplines. Despite their widespread use, there are few publications that focus on elucidating the biological pathways involved during the processing of base editor intermediates. Because base editors introduce unique types of DNA damage products (a U•G mismatch with a DNA backbone nick for CBEs, and an I•T mismatch with a DNA backbone nick for ABEs) to facilitate genome editing, a deep understanding of the DNA damage repair pathways that facilitate or impede base editing represents an important aspect for the further expansion and improvement of the technologies. Here, we first review canonical deoxyuridine, deoxyinosine, and single-stranded break repair. Then, we discuss how interactions among these different repair processes can lead to different base editing outcomes. Through this review, we hope to promote thoughtful discussions on the DNA repair mechanisms of base editing, as well as help researchers in the improvement of the current base editors and the development of new base editors.

Base Editing in Human Cells to Produce Single-Nucleotide-Variant Clonal Cell Lines.

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.