Improved design and delivery of gene-editing tools boost precise DNA changes using CRISPR/Cas9

Targeted integration efficiency of up to 56% was achieved for genetic elements in HEK293 cells.

• Precise genome editing rates of up to 45% were observed in induced pluripotent stem cells (iPSCs) using co-delivered Cas9 RNP and donor DNA.
• Integration of longer FLAG epitope tags with a restriction site was accomplished at rates of up to 50% using short double-stranded DNA oligonucleotides with 3' overhangs.
• A model suggests that donor DNAs should be designed with the change as close as possible to the cleavage site for improved integration.
• Asymmetric single-stranded donor molecules with specific lengths of homology arms are favored for small changes like SNPs or short insertions.
• For larger insertions, double-stranded DNA donors with protruding 3' homology arms of 30 bases may enhance integration efficiency.
• Phosphorothioate modifications at the ends of donor DNA can improve editing efficiency in both small and large insertions.

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