Person:

Liu, David

Base editors are widely used engineered proteins that introduce targeted point mutations into DNA without creating double-stranded DNA breaks. The application of base editors to study and treat genetic diseases is dependent on their in vivo delivery into relevant cell types. Delivery by adeno-associated virus (AAV), a clinically validated delivery method, poses special challenges because the size of base editors exceeds the AAV packaging limit. Here we describe the development and application of in vivo delivery methods for both cytosine base editors (CBEs) and adenine base editors (ABEs). Dual AAVs each provide one half of the editor and trans-splicing inteins reconstitute full base editor activity, circumventing the AAV packaging limit. We optimized each AAV component to greatly improve editing efficiency. The resulting AAVs enable in vivo base editing for the first time in mouse brain, retina, and heart, as well as the most efficient base editing to date in muscle and liver, with therapeutically relevant efficiencies at viral dosages known to be tolerated in humans. A single intravenous injection of split CBE in PHP.eB AAV resulted in editing of up to 59% of unsorted mouse cortical tissue. Intravenous injection of split CBE or ABE in AAV9 mediated up to 38%, 20%, and 9% base editing in unsorted mouse liver, heart, and skeletal muscle, respectively. Subretinal and intracerebroventricular injections of split CBE and ABE in Anc80, PHP.B/PHP.eB, and AAV9 viruses mediated mouse retina and brain editing in up to 38% and 50% of unsorted cells, respectively. We applied this system to directly correct in mouse brain tissue a mutation that causes the neurodegenerative ataxia Niemann-Pick disease type C (NPC), slowing neurodegeneration and increasing lifespan consistent with expectations based on mosaic NPC mice. These findings establish a broadly useful AAV platform for the efficient introduction of targeted point mutations into multiple tissues of therapeutic interest for which in vivo base editing has not been previously reported.

The all-protein cytosine base editor DdCBE uses TALE proteins and a double-stranded DNA-specific cytidine deaminase (DddA) to mediate targeted C•G-to-T•A editing. To improve editing efficiency and overcome the strict TC sequence-context constraint of DddA, we used phage-assisted non-continuous and continuous evolution to evolve DddA variants with improved activity and expanded targeting scope. Compared to canonical DdCBEs, base editors with evolved DddA6 improved mitochondrial DNA (mtDNA) editing efficiencies at TC by 3.3-fold on average. DdCBEs containing evolved DddA11 offered a broadened HC (H = A, C or T) sequence compatibility for both mitochondrial and nuclear base editing, increasing average editing efficiencies at AC and CC targets from less than 10% for canonical DdCBE to 15–30% and up to 50% in cell populations sorted to express both halves of DdCBE. We used these evolved DdCBEs to efficiently install disease-associated mtDNA mutations in human cells at non-TC target sites. DddA6 and DddA11 substantially increase the effectiveness and applicability of all-protein base editing.

Base editing requires that the target sequence satisfy the protospacer adjacent motif requirement of the Cas9 domain and that the target nucleotide be located within the editing window of the base editor. To increase the targeting scope of base editors, we engineered six optimized adenine base editors (ABEmax variants) that use SpCas9 variants compatible with non-NGG protospacer adjacent motifs. To increase the range of target bases that can be modified within the protospacer, we use circularly permuted Cas9 variants to produce four cytosine and four adenine base editors with an editing window expanded from ~4–5 nucleotides to up to ~8–9 nucleotides and reduced byproduct formation. This set of base editors improves the targeting scope of cytosine and adenine base editing.

Prime editing enables the installation of virtually any combination of point mutations, small insertions or small deletions in the DNA of living cells. A prime editing guide RNA (pegRNA) directs the prime editor protein to the targeted locus and also encodes the desired edit. Here we show that degradation of the 3′ region of the pegRNA that contains the reverse transcriptase template and the primer binding site can poison the activity of prime editing systems, impeding editing efficiency. We incorporated structured RNA motifs to the 3′ terminus of pegRNAs that enhance their stability and prevent degradation of the 3′ extension. The resulting engineered pegRNAs (epegRNAs) improve prime editing efficiency 3–4-fold in HeLa, U2OS and K562 cells and in primary human fibroblasts without increasing off-target editing activity. We optimized the choice of 3′ structural motif and developed pegLIT, a computational tool to identify non-interfering nucleotide linkers between pegRNAs and 3′ motifs. Finally, we showed that epegRNAs enhance the efficiency of the installation or correction of disease-relevant mutations.

The targeted deletion, replacement, integration or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically require double-strand DNA breaks (DSBs) that lead to undesired consequences, including uncontrolled indel mixtures and chromosomal abnormalities. Here we describe twin prime editing (twinPE), a DSB-independent method that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites. The two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the prime-editor-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled targeted integration of gene-sized DNA plasmids (>5,000 bp) and targeted sequence inversions of 40 kb in human cells. TwinPE expands the capabilities of precision gene editing and might synergize with other tools for the correction or complementation of large or complex human pathogenic alleles.

N6-methyladenosine (m6A) is the most widespread internal mRNA modification in humans. Despite recent progress in understanding the biological roles of m6A, the inability to install m6A site-specifically in individual transcripts has hampered efforts to establish causal relationships between the presence of a specific m6A and phenotypic outcomes. Here we demonstrate that nucleus-localized dCas13 fusions with a truncated METTL3 methyltransferase domain and cytoplasm-localized fusions with a modified METTL3:METTL14 methyltransferase complex can direct site-specific m6A incorporation in distinct cellular compartments, with the former fusion protein having particularly low off-target activity. Independent cellular assays across multiple sites confirm that this targeted RNA methylation (TRM) system mediates efficient m6A installation in endogenous RNA transcripts with high specificity. Finally, we show that TRM can induce m6A-mediated changes to transcript abundance and alternative splicing. These findings establish TRM as a tool for targeted epitranscriptome engineering to help reveal the effect of individual m6A modifications and dissect their functional roles.

The viral delivery of base editors has been complicated by their size and by the limited packaging capacity of adeno-associated viruses (AAVs). Typically, dual-AAV approaches based on trans-splicing inteins have been used. Here we show that, compared with dual-AAV systems, AAVs with size-optimized genomes incorporating compact adenine base editors (ABEs) enable efficient editing in mice at similar or lower doses. Single-AAV-encoded ABEs retro-orbitally injected in mice led to editing efficiencies in liver (66%), heart (33%) and muscle (22%) tissues that were up to 2.5-fold those of dual-AAV ABE8e, and to a 93% knockdown (on average) of human PCSK9 and of mouse Pcsk9 and Angptl3 in circulation, concomitant with substantial reductions of plasma cholesterol and triglycerides. Moreover, three size-minimized ABE8e variants, each compatible with single-AAV delivery, collectively offer compatibility with protospacer-adjacent motifs for editing approximately 82% of the adenines in the human genome. ABEs encoded within single AAVs will facilitate research and therapeutic applications of base editing by simplifying AAV production and characterization, and by reducing the dose required for the desired level of editing.