Versatile Applications of <scp>CRISPR</scp> ‐Based Programmable T‐ <scp>DNA</scp> Integration in Plants

Agrobacterium-mediated T-DNA integration into plant genomes represents a cornerstone for transgenic expression in plant basic research and synthetic biology. However, random T-DNA integration can disrupt essential endogenous genes or compromise transgene expression, stressing the need for targeted integration strategies. Here we explored CRISPR-aided targeted T-DNA integration (CRISTTIN) in Arabidopsis, leveraging CRISPR-induced double-strand breaks (DSBs) to facilitate precise T-DNA insertion. Contrary to our initial hypothesis, conventional Cas9 outperformed a designed Cas9-adaptor fusion nuclease that may recruit Agrobacterium VirD2/T-DNA complexes to DSB sites via the adaptor-VirD2 interaction. Using Cas9-based CRISTTIN, we streamlined the parallel generation of FERONIA null alleles and in-locus complementation alleles expressing a wild-type or mutated gene. This enabled phenotypic comparisons under identical genomic contexts and significantly accelerated gene characterisation and critical residue identification. Additionally, CRISTTIN was employed to simultaneously knockout AGAMOUS and in-locus integrate a RUBY reporter, yielding plants with pink double-petaled flowers. CRISTTIN also enabled site-specific insertion of 35S enhancers for transcriptional upregulation of adjacent genes or reporter constructs for promoter activity monitoring. CRISTTIN's effectiveness was further validated in rice. These results demonstrated CRISTTIN as a versatile tool for gene functional studies and precise control of transgene expression in plants.