CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK ( L ong dsDNA with 3′- O verhangs mediated C RISPR K nock-in), by utilizing specially designed 3′-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3′-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.

The end modification using fluorophore dyes or quenchers to or close to the toehold domain can significantly modulate the kinetics of toehold-mediated strand displacement reaction (TMSDR) by almost two orders...