Accurate insertion of large DNA pieces using prime editing guided by donor DNA matching

An efficiency of up to 57.8% in replacing endogenous sequences with a 2.9-kb donor DNA fragment was achieved in human HEK293T cells.

• The prime assembly (PA) tool allows for megabase-scale genomic excision and kilobase-scale donor insertion.
• PA can utilize DNA plasmids and linear double-stranded DNA donors ranging from 1.0 to 6.5 kb in size.
• Integration accuracy for PA fragments exceeds 90%.
• Chimeric antigen receptor integration using PA in primary human T cells reached up to 28.1% efficiency.
• When PA with a GFP donor was injected into mice, an average integration efficiency of 4.3% was observed in GFP-positive liver cells.

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