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. 2021 Oct 19;10:e72216. doi: 10.7554/eLife.72216

Figure 2. End-modified donors promote homology-directed repair (HDR) at endogenous loci in mammalian cell cultures.

(A) Schematic representation of the 5′-modified donor design for enhanced green fluorescent protein (eGFP) insertion and fluorescence-activated cell sorting (FACS) is shown. Efficacy of eGFP integration at (B) TOMM20 and (C) GAPDH (D) Sec61B loci in HEK293T cells using unmodified, triethylene glycol (TEG) or 2′-O-methyl (2′OMe)-RNA::triethylene glycol (TEG)-modified donors are plotted as percentage of GFP+ cells. Efficacy of eGFP integration at the (E) TOMM20 locus in human foreskin fibroblast (HFF) (747 bp knock-in with ~1 kb homology arms) and (F) Gapdh locus in Chinese hamster ovary (CHO) (1635 bp knock-in with ~800 bp homology arms) cells using double-stranded DNA (dsDNA) (500 ng) donors with and without 2′OMe-RNA::TEG modifications at the 5′-ends. (G) Schematic representation of the dsDNA donor design used for quantification with deep sequencing is shown. (H) Illumina sequencing reads with precise knock-in are plotted for dsDNA donors with 90 bp homology arms at EMX1 locus in HEK293T cells. Mean ± s.d. for at least three independent replicates are plotted. p-Values were calculated using one-way ANOVA and in all cases end-modified donors were compared to unmodified donor unless indicated otherwise (Tukey’s multiple comparisons test; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant).

Figure 2.

Figure 2—figure supplement 1. RNA::TEG (triethylene glycol) donors with short (90 bp) homology arms are more potent than unmodified donors at EMX1 locus.

Figure 2—figure supplement 1.

Fraction of precise reads is plotted as percentage of total Illumina reads obtained at various amounts of double-stranded DNA (dsDNA) donors into K562 cells. Cas9 ribonucleoproteins (RNPs) and dsDNA donors were nucleofected into K562 cells and harvested after 3 days.