Most genetic variants that contribute to disease 1 are challenging to correct efficiently and without excess byproducts 2-5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed >175 edits in human cells including targeted insertions, deletions, and all 12 types of point mutations without requiring double-strand breaks or donor DNA templates. We applied prime editing in human cells to correct efficiently and with few byproducts the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA), to install a protective transversion in PRNP, and to precisely insert various

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Base editors enable targeted single-nucleotide conversion in genomic DNA. Here we show that expression levels are a bottleneck in base editing efficiency. We optimize cytidine (BE4) and adenine (ABE7.10) base editors by modification of nuclear localization signals and codon usage, and ancestral reconstruction of the deaminase component. The resulting BE4max, AncBE4max, and ABEmax editors correct pathogenic SNPs with substantially increased efficiency in a variety of mammalian cell types.

Applications of adenine base editors (ABEs) have been constrained by the limited compatibility of the deoxyadenosine deaminase component with Cas homologs other than SpCas9. We evolved the deaminase component of ABE7.10 using phage-assisted non-continuous and continuous evolution (PANCE and PACE), resulting in ABE8e. ABE8e contains eight additional mutations that increase activity (k app ) 590-fold compared with ABE7.10. ABE8e offers substantially improved editing efficiencies when paired with a variety of Cas9 or Cas12 homologs. ABE8e is more processive than ABE7.10, which could benefit screening, disrupting regulatory regions and multiplex base editing applications. A modest increase in Cas9-dependent and -independent DNA off-target editing, and in transcriptome-wide RNA off-target editing can be ameliorated by introducing additional mutations in the TadA-8e domain. Finally, we show that ABE8e can efficiently edit natural mutations in a GATA1 binding site in the BCL11A enhancer or the HBG promoter in human cells, targets which were poorly edited with ABE7.10. ABE8e broadens the effectiveness and applicability of adenine base editing.

Probing base editing outcomes leads to new C:G to T:A base editors with greater efficiency and product purity, and fewer indels.

The success of base editors for the study and treatment of genetic diseases depends on the ability to deliver them in vivo to the relevant cell types. Delivery via adeno-associated viruses (AAVs) is limited by AAV-packaging capacity, which precludes the use of full-length base editors. Here, we report the application of dual AAVs for the delivery of split cytosine and adenine base editors that are then reconstituted by trans-splicing inteins. Optimized dual AAVs enable in vivo base editing at therapeutically relevant efficiencies and dosages in the mouse brain (up to 59% of unsorted cortical tissue), liver (38%), retina (38%), heart (20%) and skeletal muscle (9%). We also show that base editing corrects, in mouse brain tissue, a mutation that causes Niemann-Pick disease type C (a neurodegenerative ataxia), slowing down neurodegeneration and increasing the animals' Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Summary Hutchinson-Gilford progeria syndrome (HGPS) is typically caused by a dominant-negative C•G-to-T•A mutation (c.1824 C>T, G608G) in LMNA , the nuclear lamin A gene. This mutation causes RNA mis-splicing that produces progerin, a toxic protein that induces rapid aging and shortens lifespan to ~14 years 1 – 4 . Adenine base editors (ABEs) perform targeted A•T-to-G•C base pair conversion with minimal byproducts and without requiring double-strand DNA breaks or donor DNA templates 5 , 6 . Here, we describe the use of an ABE to directly correct the pathogenic HGPS mutation in cultured progeria patient-derived fibroblasts and in a mouse model of HGPS. Lentiviral delivery of ABE to patient-derived fibroblasts results in ~90% correction of the pathogenic allele, mitigation of RNA mis-splicing, reduced progerin levels, and correction of nuclear abnormalities. Unbiased off-target DNA and RNA analysis did not detect off-target editing activity in treated patient-derived fibroblasts. In transgenic mice homozygous for the human LMNA c.1824 C>T allele, a single retro-orbital injection of adeno-associated virus 9 (AAV9) encoding the ABE resulted in substantial, durable correction of the pathogenic mutation (~20-60% across various organs 6 months post-injection), restoration of normal RNA splicing, and reduction of progerin protein. In vivo base editing rescued vascular pathology, preserving vascular smooth muscle cell counts and preventing adventitial fibrosis. A single ABE AAV9 injection at P14 improved animal vitality and greatly extended median lifespan from 215 to 510 days. These findings support the potential of in vivo base editing to treat HGPS, and other genetic diseases, by directly correcting the root cause of disease.

We used BE-PACE to evolve new cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing GC, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width, and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.Genome editing has revolutionized the life sciences and entered clinical trials to treat genetic diseases. 1 The use of programmable nucleases to generate double-stranded DNA breaks (DSBs) followed by homology-directed repair can introduce a wide variety of modifications but is inefficient in non-dividing cells, and is typically accompanied by an excess of unwanted insertions and deletions (indels), translocations, or other chromosomal rearrangements. 2 Base editing directly modifies target DNA bases in living cells and has become widely used to correct or install point mutations in organisms ranging from bacteria to human embryos. 3 Base editors use a catalytically impaired Cas9 to open a single-stranded DNA loop at a specified genomic site (Fig. 1a). Bases within the editing window (typically ~5 nt wide) in this region are modified by a tethered base-modification enzyme that only accepts single-stranded DNA.