Base editing: advances and therapeutic opportunities

Abstract

Base editing - the introduction of single-nucleotide variants (SNVs) into DNA or RNA in living cells - is one of the most recent advances in the field of genome editing. As around half of known pathogenic genetic variants are due to SNVs, base editing holds great potential for the treatment of numerous genetic diseases, through either temporary RNA or permanent DNA base alterations. Recent advances in the specificity, efficiency, precision and delivery of DNA and RNA base editors are revealing exciting therapeutic opportunities for these technologies. We expect the correction of single point mutations will be a major focus of future precision medicine.

Fig. 1 |. General overview of DNA base editing technologies.

a | Cytosine base editor (CBE) mechanism. Principle components of the CBE are designated in coloured text boxes. If uracil glycosylase inhibitor (UGI) is present (an optional component), it will ‘protect’ the U•G intermediate from excision by uracil DNA glycosylase (UDG) to boost efficiency of the final base-edited DNA outcome. The nickase version of Cas9 (Cas9n) nicks the top strand (red arrow) whereas the cytidine deaminase converts cytosine (red) to uracil (green). Ultimate conversion of a C•G to T•A base pair is achieved through the outlined steps. b | The adenine base editor (ABE) mechanism is similar to that of CBE, without possible inclusion of a UGI domain in the ABE architecture. Through ABE-mediated editing, an A•T to G•C base pair conversion is achieved via an inosine-containing intermediate. gRNA, guide RNA; PAM, protospacer adjacent motif; target A, ABE desired base substrate; target C, CBE desired base substrate. Part a adapted from REF., Springer Nature Limited. Part b adapted from REF., Springer Nature Limited.

Fig. 2 |. General overview of RNA base editing technologies.

a | Antisense oligonucleotide (ASO)-mediated A-to-I RNA base editing with an engineered adenosine deaminase, using the λN–BoxB construct as an example. The catalytic domain of ADAR2 (ADAR2_DD; yellow) is fused to multiple copies of the λN coat protein (green). The ASO guide RNA (gRNA; green strand) is engineered to base pair with the target mRNA (blue strand) and contain multiple BoxB hairpins to recruit the λN peptide to the mRNA of interest. An induced A•C mismatch between the target RNA and the ASO is employed to achieve targeted adenosine deamination on mRNA by ADAR2. b | ASO-mediated A-to-I RNA base editing with an endogenous adenosine deaminase, using RESTORE (recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing) as an example. Endogenous ADAR1 comprises a catalytic domain and two double-stranded RNA binding domains (dsRBDs; blue). The engineered ASO gRNA (green strand) consists of a specificity domain (the 3′ end, which binds to the target mRNA of interest (blue strand) through Watson–Crick–Franklin base pairing) and an ADAR-recruiting domain (the 5′ end, which comprises the natural substrate of the dsRBD), which recruits endogenous ADAR1 to a target mRNA of interest, where an induced A•C mismatch between the target RNA and the ASO directs the catalytic domain of ADAR1 for targeted adenosine deamination. c | A-to-I RNA base editing through REPAIR (RNA editing for programmable A-to-I replacement). An induced A•C mismatch between the target mRNA (blue strand) and the gRNA (green strand) of Cas13b (blue) is employed to achieve targeted adenosine deamination on mRNA by ADAR2. d | C-to-U RNA base editing through RESCUE (RNA editing for specific C-to-U exchange). An induced C•C or C•U mismatch between the target mRNA (blue strand) and the gRNA (green strand) of Cas13b (blue) is employed to achieve targeted cytosine deamination on mRNA by a mutant version of ADAR2. ADAR, adenosine deaminase acting on RNA; dCas, catalytically dead Cas enzyme; e, enhanced; target A, REPAIR desired base substrate; target C, RESCUE desired base substrate. Part a adapted with permission from REF., OUP. Part b adapted from REF., Springer Nature Limited. Part c adapted with permission from REF., AAAS. Part d adapted with permission from REF..

Fig. 3 |. DNA base editor and protospacer design scheme.

a | Construct maps of basic cytosine base editor (CBE) and adenine base editor (ABE) architectures. In the CBE architecture (top), solid line components make up the basis for the fourth-generation CBE, BE4, whereas dotted line components (bipartite nuclear localization signal (bpNLS); green) can be optionally added, to produce BE4max. The amino-terminal bpNLS* component is FLAG-tagged (yellow haze). In the ABE architecture (bottom), all solid line and dotted line components make up the basis for ABE7.10; the dotted lined components (wtTadA (orange) and one of the two 32-amino-acid (aa) linkers (grey)) are optional, and removal of these components results in a monomeric ABE construct with no reduction in on-target efficiency. For both CBE and ABE architectures, use of an appropriate nickase Cas variant is only possible with Cas9 (Cas9n; blue). b | Activity windows of base editors with the basic architecture from part a with the indicated Cas proteins (Streptococcus pyogenes Cas9 (SpCas9; blue), Staphylococcus aureus Cas9 (SaCas9; green) and Cas12a (purple)). Protospacer adjacent motifs (PAMs) associated with each Cas enzyme are listed. Base editor activity windows are shown over the 20-nucleotide protospacer sequence (corresponding coloured box outlines). c | Simplified workflow of protospacer and DNA base editor design strategy for user-defined adaptation. Once a protospacer, Cas enzyme variant and basic architecture are chosen, modifications can be incorporated for each specific application according to TABLE 1. CP, circular permutant; dCas, catalytically dead Cas enzyme; rAPOBEC1, APOBEC1 from Rattus norvegicus; TadA*, mutated TadA (contains ABE7.10 or ABE8 mutations as indicated); target A, ABE desired base substrate; target C, CBE desired base substrate; UGI, uracil glycosylase inhibitor; wt, wild-type.

Fig. 4 |. Base editor delivery strategies.

Localized nanoparticle injection (red), systemic viral administration (blue) and ex vivo electroporation (green) collectively make up the three primary means to potentially administer base editor therapeutics. Each methodology is shown with the corresponding range of delivery vehicles or target cells. Red and green panels show base editor delivery through nucleic acid (i.e. mRNA or DNA) or RNP format. *, suite of virus options; **, suite of nanoparticle options; ***, suite of cell target options; CAR, chimeric antigen receptor; gRNA, guide RNA; HSC, haematopoietic stem cell; RNP, ribonucleoprotein complex.