Engineered CRISPR prime editors with compact, untethered reverse transcriptases

Nature Biotechnology, Год журнала: 2022, Номер 41(3), С. 337 - 343

Опубликована: Сен. 26, 2022

Язык: Английский

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Peptide fusion improves prime editing efficiency

Nature Communications, Год журнала: 2022, Номер 13(1)

Опубликована: Июнь 18, 2022

Abstract Prime editing enables search-and-replace genome but is limited by low efficiency. We present a high-throughput approach, the Peptide Self-Editing sequencing assay (PepSEq), to measure how fusion of 12,000 85-amino acid peptides influences prime show that peptide can enhance editing, prime-enhancing combine productively, and top dual peptide-prime editor increases significantly in multiple cell lines across dozens target sites. Top function increasing translation efficiency serve as broadly useful tools improve

Язык: Английский

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Adenine Base Editing In Vivo with a Single Adeno-Associated Virus Vector

GEN Biotechnology, Год журнала: 2022, Номер 1(3), С. 285 - 299

Опубликована: Июнь 1, 2022

Base editors (BEs) have opened new avenues for the treatment of genetic diseases. However, advances in delivery approaches are needed to enable disease targeting a broad range tissues and cell types. Adeno-associated virus (AAV) vectors remain one most promising vehicles gene therapies. Currently, BE/guide combinations their promoters exceed packaging limit (∼5 kb) AAVs. Dual-AAV strategies often require high viral doses that impose safety concerns. In this study, we engineered an adenine base editor (ABE) using compact Cas9 from Neisseria meningitidis (Nme2Cas9). Compared with well-characterized Streptococcus pyogenes Cas9-containing ABEs, ABEs Nme2Cas9 (Nme2-ABE) possess distinct protospacer adjacent motif (N4CC) editing window, exhibit fewer off-target effects, can efficiently install therapeutically relevant mutations both human mouse genomes. Importantly, show vivo Nme2-ABE its guide RNA by single AAV vector edit genomic loci revert mutation phenotype adult model tyrosinemia. We anticipate Nme2-ABE, virtue size range, will therapeutic applications improved efficacy due part single-vector system.

Язык: Английский

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Template-jumping prime editing enables large insertion and exon rewriting in vivo

Nature Communications, Год журнала: 2023, Номер 14(1)

Опубликована: Июнь 8, 2023

Targeted insertion of large DNA fragments holds promise for genome engineering and gene therapy. Prime editing (PE) effectively inserts short (<50 bp) sequences. Employing paired prime guide RNAs (pegRNAs) has enabled PE to better mediate relatively insertions in vitro, but the efficiency larger (>400 remains low vivo application not been demonstrated. Inspired by efficient genomic mechanism retrotransposons, we develop a template-jumping (TJ) approach using single pegRNA. TJ-pegRNA harbors sequence as well two primer binding sites (PBSs), with one PBS matching nicking sgRNA site. TJ-PE precisely 200 bp 500 up 50.5 11.4% efficiency, respectively, enables GFP (~800 expression cells. We transcribe split circular TJ-petRNA vitro via permuted group I catalytic intron non-viral delivery Finally, demonstrate that can rewrite an exon liver tyrosinemia mice reverse disease phenotype. potential insert without double-stranded breaks facilitate mutation hotspot rewriting vivo.

Язык: Английский

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A prime editor mouse to model a broad spectrum of somatic mutations in vivo

Nature Biotechnology, Год журнала: 2023, Номер unknown

Опубликована: Май 11, 2023

Язык: Английский

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