The Wild-Type tRNA Adenosine Deaminase Enzyme TadA Is Capable of Sequence-Specific DNA Base Editing.

Base editors are genome editing tools that enable site-specific base conversions through the chemical modification of nucleobases in DNA. Adenine base editors (ABEs) convert A ⋅ T to G ⋅ C base pairs in DNA by using an adenosine deaminase enzyme to modify target adenosines to inosine intermediates. Due to the lack of a naturally occurring adenosine deaminase that can modify DNA, ABEs were evolved from a tRNA-deaminating enzyme, TadA. Previous experiments with an ABE comprising a wild-type (wt) TadA showed no detectable activity on DNA, and directed evolution was therefore required to enable this enzyme to accept DNA as a substrate. Here we show that wtTadA can perform base editing in DNA in both bacterial and mammalian cells, with a strict sequence motif requirement of TAC. We leveraged this discovery to optimize a reporter assay to detect base editing levels as low as 0.01 %. Finally, we used this assay along with molecular dynamics simulations of full ABE:DNA complexes to better understand how the sequence recognition of mutant TadA variants change as they accumulate mutations to better edit DNA substrates.

Genome, Epigenome, and Transcriptome Editing via Chemical Modification of Nucleobases in Living Cells.

Base editors are tools that chemically modify the nucleobases of DNA and RNA in a programmable manner, allowing for genome, epigenome, and transcriptome editing in live cells. These tools can be used to introduce specific base transitions in DNA or RNA, manipulate methylation patterns in the epigenome, and create genetically encoded libraries in target genes. These various functions can be used to modulate every aspect of the central dogma. The efficiency and precision of base editors makes them useful in both basic research and the development of new therapies. Here we describe currently available base editors and the ways that they can be used to better understand and manipulate different aspects of the central dogma.

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.

A Genetically Encoded Fluorescent Biosensor for Intracellular Measurement of Malonyl-CoA.

Malonyl-CoA is the essential building block of fatty acids and regulates cell function through protein malonylation and allosteric regulation of signaling networks. Accordingly, the production and use of malonyl-CoA is finely tuned by the cellular energy status. Most studies of malonyl-CoA dynamics rely on bulk approaches that take only a snapshot of the average metabolic state of a population of cells, missing out on dynamic changes in malonyl-CoA and fatty acid biosynthesis that could be occurring within a single cell. To overcome this limitation, we have developed a genetically encoded fluorescent protein-based biosensor for malonyl-CoA that can be used to capture malonyl-CoA dynamics in single cells. This biosensor, termed Malibu (malonyl-CoA intracellular biosensor to understand dynamics), exhibits an excitation-ratiometric change in response to malonyl-CoA binding. We first used Malibu to monitor malonyl-CoA dynamics during inhibition of fatty acid biosynthesis using cerulenin in E. coli, observing an increase in Malibu response in a time- and dose-dependent manner. In HeLa cells, we used Malibu to monitor the impact of fatty acid biosynthesis inhibition on malonyl-CoA dynamics in single cells, finding that two inhibitors of fatty acid biosynthesis, cerulenin and orlistat, which inhibit different steps of fatty acid biosynthesis, increase malonyl-CoA levels. Altogether, we have developed a new genetically encoded biosensor for malonyl-CoA, which can be used to sensitively study malonyl-CoA dynamics in single cells, providing an unparalleled view into fatty acid biosynthesis.

Expanded palette of RNA base editors for comprehensive RBP-RNA interactome studies.

RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To broaden the repertoire of rBEs suitable for editing-based RBP-RNA interaction studies, we have devised experimental and computational assays in a framework called PRINTER (protein-RNA interaction-based triaging of enzymes that edit RNA) to assess over thirty A-to-I and C-to-U rBEs, allowing us to identify rBEs that expand the characterization of binding patterns for both sequence-specific and broad-binding RBPs. We also propose specific rBEs suitable for dual-RBP applications. We show that the choice between single or multiple rBEs to fuse with a given RBP or pair of RBPs hinges on the editing biases of the rBEs and the binding preferences of the RBPs themselves. We believe our study streamlines and enhances the selection of rBEs for the next generation of RBP-RNA target discovery.

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

The flexibility and precision of CRISPR-Cas9 and related technologies have made these genome editing tools increasingly popular in agriculture, medicine, and basic science research over the past decade. Genome editing will continue to be relevant and utilized across diverse scientific fields in the future. Given this, students should be introduced to genome editing technologies and encouraged to consider their ethical implications early on in pre-college biology curricula. Furthermore, instruction on this topic presents an opportunity to create partnerships between researchers and educators at the K-12 levels that can strengthen student engagement in science, technology, engineering, and mathematics (STEM). To this end, we present a three-day student-centered learning program to introduce high school students to genome editing technologies through a hands-on base editing experiment in E. coli , accompanied by a relevant background lecture and facilitated ethics discussion. This unique partnership aims to educate students and provides a framework for research institutions to implement genome editing outreach programs at local high schools.

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

The flexibility and precision of CRISPR-Cas9 and related technologies have made these genome editing tools increasingly popular in agriculture, medicine, and basic science research for the past decade. Genome editing will continue to be relevant and utilized across diverse scientific fields in the future. Given this, students should be introduced to genome editing technologies and encouraged to consider their ethical implications early on in precollege biology curricula. Furthermore, instruction on this topic presents an opportunity to create partnerships between researchers and educators at the K-12 levels that can strengthen student engagement in science, technology, engineering, and mathematics. To this end, we present a 3-day student-centered learning program to introduce high school students to genome editing technologies through a hands-on base editing experiment in Escherichia coli, accompanied by a relevant background lecture and facilitated ethics discussion. This unique partnership aims to educate students and provides a framework for research institutions to implement genome editing outreach programs at local high schools. We have included all requisite materials, including lecture slides, worksheets, experimental protocols, and suggestions on active learning strategies for others to reproduce our program with their local communities.

Expanded palette of RNA base editors for comprehensive RBP-RNA interactome studies.

RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To broaden the repertoire of rBEs suitable for editing-based RBP-RNA interaction studies, we have devised experimental and computational assays in a framework called PRINTER (protein-RNA interaction-based triaging of enzymes that edit RNA) to assess over thirty A-to-I and C-to-U rBEs, allowing us to identify rBEs that expand the characterization of binding patterns for both sequence-specific and broad-binding RBPs. We also propose specific rBEs suitable for dual-RBP applications. We show that the choice between single or multiple rBEs to fuse with a given RBP or pair of RBPs hinges on the editing biases of the rBEs and the binding preferences of the RBPs themselves. We believe our study streamlines and enhances the selection of rBEs for the next generation of RBP-RNA target discovery.

Combined Theoretical, Bioinformatic, and Biochemical Analyses of RNA Editing by Adenine Base Editors.

Adenine base editors (ABEs) have been subjected to multiple rounds of mutagenesis with the goal of optimizing their function as efficient and precise genome editing agents. Despite an ever-expanding data set of ABE mutants and their corresponding DNA or RNA-editing activity, the molecular mechanisms defining these changes remain to be elucidated. In this study, we provide a systematic interpretation of the nature of these mutations using an entropy-based classification model that relies on evolutionary data from extant protein sequences. Using this model in conjunction with experimental analyses, we identify two previously reported mutations that form an epistatic pair in the RNA-editing functional landscape of ABEs. Molecular dynamics simulations reveal the atomistic details of how these two mutations affect substrate-binding and catalytic activity, via both individual and cooperative effects, hence providing insights into the mechanisms through which these two mutations are epistatically coupled.