Donor-complementary prime editing enables precise kilobase and library-compatible DNA insertions.

Precise genomic integration of large DNA fragments by donor-directed annealing using prime editing.

A programmable gene replacement tool, named prime assembly (PA), which adapts prime editors to produce one or two pairs of 3'-flaps on both the genome and donor DNA, allowing megabase-scale genomic excision and/or kilobase-scale donor insertion at the gene of interest.

Efficient and precise programmable DNA knock-in without double-strand breaks

Kilobase-scale nickase-targeting (KNIT) editing enables single-nick-based DNA insertion across genomic loci and cell types, supporting insertion of DNA fragments exceeding 10 kb, with high efficiency and minimal unwanted off-target effects.

A self-iterative orthogonal base-editing platform enables multiplex N-to-N diversification and genome-scale functional screening in Escherichia coli

Abstract Base editing enables precise genome modification without double-strand breaks but remains limited by narrow editing windows, DNA repair pathway biases, and restricted nucleotide diversity. Here, we report MUTATOR, a MUlTiplexAble and self-iTerative ORthogonal base-editing platform that enables N-to-N diversification in Escherichia coli. MUTATOR combines CWBE and ABE with iterative editing on two complementary DNA strands, thereby overcoming endogenous DNA repair constraints and expanding A-to-N and C-to-N editing outcomes across both strands. This strategy substantially expands accessible nucleotide outcomes, codon variants, and amino-acid diversity within existing editing windows relative to conventional editors. Using four gRNAs, MUTATOR facilitated four-site editing of ompR, generating 84 distinct amino-acid combinations and 252 codon combinations, with the synonymous OmpR_P160P variant increasing isobutanol production by up to 56.2%. We further applied MUTATOR to a 151-gene library encompassing transcriptional regulators, translation factors, DNA repair proteins, ribosomal components, and NAD(P)H-associated metabolic genes, identifying single and combinatorial mutations that markedly enhanced cell growth and ethanol utilization when ethanol was used as the sole carbon source. Together, these results establish MUTATOR as a broadly applicable platform for genome-wide diversification, functional dissection, and rapid engineering of industrial microbial chassis.

Methods for Applying Prime Editing and Inverse Prime Editing in Mammalian Cell Culture.

From Double-Strand Breaks to Precision Edits: A Comparative Review of Modern Genome Editing Tools

This review compares Cas9-mediated homology-directed repair (HDR) with generations of cytosine base editors (CBE1–CBE3), adenine base editors (ABE1-ABE7), and prime editors (PE1–PE3b), focusing on their mechanistic distinctions, efficiencies, delivery challenges, and therapeutic applications.

High-diversity base mutagenesis via simultaneous adenine, cytosine and guanine editing

Base editors hold great promise in endogenous mutagenesis for genetic screening. However, the development of base editors that induce saturated multi-base conversions with diverse mutation spectrum is challenging. Here, we develop triple base editors (smACGs) that simultaneously mutagenize adenine, cytosine, and guanine within the same allele. Through screening and embedding engineered deaminase and alkyladenine DNA glycosylase variants in Cas9 structure, smACGmax is generated to catalyze robust triple-base conversion efficiencies of up to 41% across varied sequence contexts while maintaining low RNA off-target effects compared to previous dual-base editors. We apply smACGmax to enable high coverage (94%) of targeted HBEGF mutagenesis that identified diphtheria toxin-resistant mutations and to dissect SF3B1 variants with alternative splicing specificity via complex single, double, and triple base conversion screening. smACGmax expands base conversion capability from single and double substrates to trinucleotide level, which facilitates the generation of high-diversity and complex genetic variants, providing a useful platform for mutagenesis-based application. Broad-spectrum base mutagenesis at the same endogenous loci with base editors remains a challenge. Here, the authors developed smACGmax to catalyze efficient multi-base conversions across adenine, cytosine and guanine, and enable high-diversity functional screening in HBEGF and SF3B1 variants.