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

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

Efficient homologous replacement and deletion of large genomic fragments through template-jumping prime editing in rice.

Homologous replacement of genomic sequences with large DNA fragments (> 100 bp) holds great potential for crop breeding, yet an efficient method to achieve such edits is lacking in plants. Here, in rice, we developed template-jumping prime editing (TJ-PE), a recently reported PE strategy for large targeted insertion, as an efficient tool for homologous replacement with DNA fragments ranging from dozens to hundreds of base pairs, and using TJ-PE, we replaced genomic fragments of up to 340 bp with homologous fragments of the same length. In addition, our TJ-PE tool also enabled precise deletion of 944- to 2024-bp fragments in rice, with efficiencies of up to 34.6% for c. 2000-bp precise deletions. Collectively, this study expands the editing scope of PE in rice and establishes TJ-PE as a generalist tool for precise deletion and replacement of large DNA fragments.

Engineering RNA-guided bridge recombinases for precise large-scale genome editing.

An RNA-guided bridge recombinase system is engineered through rational mutagenesis and AI-assisted directed evolution, enabling programmable chromosomal rearrangements in both plant and mammalian cells and achieving up to a 29.8-fold increase in activity.

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

Efficient CRISPR/Cas9-mediated homology independent sequence replacement in vivo and non-dividing primary cells

REPLACE is established as a versatile platform for homology-independent sequence replacement and as a complementary approach for genetic correction in settings where homology-directed repair (HDR), base editing (BE) or prime editing (PE) are inefficient or not applicable.

Simplifying Multiplex Genome Engineering in Saccharomyces cerevisiae with Intron-mediated Random Assembly and INtegration (RAIN).

Engineering of multi-step enzymatic pathways often involves extensive optimisation of heterologous gene expression levels and requires cloning of promoter and open reading frames (ORFs) to generate expression cassettes. We present work on a nascent method for multiplex genome engineering in Saccharomyces cerevisiae that negates the requirement for cloning of expression cassettes. Our system, Random Assembly and INtegration (RAIN), uses intron-mediated homologous recombination (HR) for random in vivo assembly of exogenous promoter and open reading frame (ORF) libraries which are combined and co-transformed in a one-pot method. The libraries include consensus homology arms which target long terminal repeat regions (LTR) of the Ty1 retrotransposon, providing over a hundred possible integration loci. In this way, our developmental system aims to negate the need for in vitro combinatorial cloning of promoters and ORFs to generate expression cassettes, simplifying in vitro DNA preparation before multiplex genome engineering. This paper presents findings from a series of experiments to demonstrate a proof of concept for the RAIN system. These include: the first reported use of intron-mediated assembly of promoters and ORFs for expression of a functional gene product; up to three markerless genomic integrations; and up to five integrations with antibiotic selection. We also present a number of innovations to improve integration efficiency during multiplex engineering in S. cerevisiae including: SGS1 gene knockout; disruption of heteroduplex rejection; modified Cas9 expression architecture; and overexpression HR genes RAD52, MRE11, and RAD59. To demonstrate how our system can be used for single transformation phenotype engineering of multiple strains, we also transformed a library of methylotrophy associated genes to generate four new strains that were able to grow on a solid minimal medium with methanol as the sole additional carbon source. Our findings contribute to the ongoing efforts to improve multiplex genome engineering tools in S. cerevisiae, and provide the foundations for further development of a novel toolbox for generating useful genetic diversity for metabolic pathway engineering.