PAMless and precise sequence replacement by gdt/Cas3 or gdt/Cas9 ribonucleoprotein

Sequence replacement is the most direct and powerful genome editing strategy. However, it remains currently an urgent need to develop protospacer adjacent motif (PAM)-less, off-target-free and simple tools for precise sequence replacement. To address this challenge, we fused a rice-derived AT-rich pincer-like elements (APE), which is composed of unique repeat-spacer array, to donor template against target sequence, thus forming guider and donor template (gdt). The donor template harbors multiple sites of DNA fragment insertion/deletion (MsDFID) which function as donor sites. APE plays as MsDFID gdt scaffold to repurpose Cas3 or Cas9 to mediate transposition of DNA fragment insertion/deletion from MsDFID donor template into genome target in E . coli , thus realizing seamless sequence replacement. These results established putative gdt/Cas3 or gdt/Cas9 ribonucleoprotein as compact genome editors which feature PAM-lessness, no observable off-target, and simplicity based on the dual role of MsDFID gdt per se as both guider and donor template. This strategy provides significant potential for precise sequence replacement in both animals and plants.