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.

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.

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.

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.