Targeted large DNA insertion in crops using large serine integrases

Overview

This research program develops a precision tool for inserting large DNA sequences into specific, pre-chosen locations in crop genomes. Unlike traditional transgenesis, which relies on random integration and produces unpredictable expression, this approach uses large serine integrases (LSIs)—enzymes that evolved to splice entire phage genomes into host DNA at specific attachment sites. By matching thousands of naturally occurring integrase specificities to accessible, single-copy safe-harbor regions in Camelina sativa and Brassica genomes, the platform enables clean, targeted insertion of sizable genetic payloads. The technology also includes a mechanism for removing vector backbone and integrase genes, leaving a reusable landing pad for sequential, modular insertions.

Technical specifications

Core mechanism:

• LSIs catalyze unidirectional insertion requiring only two short cognate sites: an approximately 40 bp attB and an approximately 50 bp attP
• Unlike Cre or Flp recombinases, LSI activity is irreversible and does not rely on host DNA repair machinery
• Thousands of LSI specificities exist in phage and mobile-element databases, providing a natural repertoire for matching desired genomic targets

Bioinformatic targeting pipeline:

• Position-weighted attB sequence matrices are built for each candidate integrase
• Camelina and Brassica genomes are scanned for high-quality attB matches
• Candidate sites are filtered to open chromatin regions at least 2 kb from any gene, in low-transposon, low-methylation areas to minimize positional silencing

Delivery and validation:

• Agrobacterium T-DNA delivery combined with in vivo T-DNA circularization
• Constructs carry selectable marker, cargo, integrase gene, and verified attP site
• Camelina transformation via floral dip; insertion confirmed by T-DNA-seq, Nanopore sequencing, and junction PCR
• On-target efficiency, off-target activity, and copy number quantified

Clean insertion and reusability:

• Backbone and integrase genes are flanked by attL and attR sites
• A second orthogonal integrase with an identified recombination directionality factor excises vector remnants
• A small attB scar remains as a landing pad for future nested insertions

Technology readiness level

The program is at an early-to-mid research stage. LSI biochemistry and mechanism are well established in the Rice laboratory. The current work focuses on bioinformatic identification of suitable integrase-genome site pairs, validation of activity in E. coli, and initial plant insertion testing in Camelina. Future validation will extend the approach to Brassica and demonstrate clean, multiplexed insertions at predefined loci. The technology is not yet commercially deployed but is advancing toward demonstrated site-specific insertion in a crop species.