Fig. 3: PE and transposase-based DNA insertion methods.
From: The evolving landscape of precise DNA insertion in plants
A Prime editing (PE)-based DNA insertion methods. This revolutionary technique employs a paired prime editing guide RNA (pegRNA) approach with forward (pegRf) and reverse (pegRr) pegRNAs. The nickase Cas9 (nCas9) protein nicks the non-target strand, while the reverse transcriptase (RT) enzyme copies genetic information from the 3′ extension of the pegRNAs, which contain the inserted nucleotides, into the nicked ends. This process forms a pair of complementary 3′ flaps that contain the desired inserted nucleotides. The annealing of these flaps allows for the seamless integration of the intended DNA sequence into the genomic site. B PrimeRoot. The paired pegRNA approach can also be used to install a recombination site (RS), such as lox66, at a desired location, typically a genomic safe harbor (GSH). Following this, the Cre recombinase, which is co-expressed with the prime editing (PE) tool or later introduced to the RS-inserted cells, facilitates strand exchanges between a circularized donor DNA carrying an inserted DNA fragment and a lox71 site. This innovative strategy enables the insertion of large DNA fragments, up to kilobases in size, in plants. C Transposase-assisted target-site integration (TATSI). In this system, a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) 9 (CRISPR-Cas9) or Cas12a protein is fused with the Pong transposase open reading frame 1 (ORF1)-ORF2 and guided by a gRNA to generate a DSB at the target site. The ORF1 binds to a mPing DNA donor, which is flanked by two 15-bp inverted repeats (indicated by green arrows) that have TTA or TAA repeats at their terminal ends, necessary for excision. The Pong transposase then excises and inserts the mPing donor (with TTA/TAA staggered ends) into the cleavage site (or within its 4-bp) created by the CRISPR-Cas protein, leaving minimal scars (usually with TTA/TAA duplication) at the junctions. This method allows for the insertion of intended DNA sequences up to several kilobases in size into the genomes of Arabidopsis and soybean using the mPing donor. Pol.: DNA polymerase. Created in BioRender https://BioRender.com/tjvvf6f.
