Custom Modified Oligos

Case Studies

Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homologydirected repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of singlestranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing.

(A) Integration activity of short ss- and dsODN donors. TALEN target sequences (in bold) and PAM sequence (in lower cases) in an intron of the PPP1R12C gene within the chromosome 19 locus are shown. Single-stranded donor oligonucleotides (ssODNs) with short homology arms, ss1s (43-mer) and ss2s and ss3s (58-mer), and the 58-bp DS3 duplex (formed by hybridization of ss3s with its complementary strand) were used to make a sequence insertion including a PvuII site at the cleavage site. A control ssODN including a PvuII site but lacking homology arms, GFPss2s-PS, was also used. Doublestranded donors (dsODNs) (different versions of DS1 and DS2, as indicated) included an XhoI site (see sequences in Tables S1 and S2 and Figure S1). dsODNs containing microhomologous ends (DS1-mhDS-PS, DS1-50 mh-PS, DS2-50 mh-PS, DS2-50 mh-PO, and DS1- 30 mh-PS) or not (DS1-blunt-PS, DS1-bluntP-PS, and DS2-50 scr-PS) were evaluated. RFLP analysis of DNA from U2OS cells treated with TALEN AAVS1 or (sgT2+Cas9) and various ssODNs (at 12 mg, except ss2s-PS at 6, 9, and 12 mg) or dsODNs (at 12 mg) is shown. The corresponding rate of PvuII or XhoI cleavage (% RFLP) and of mutations evaluated in parallel by the T7 endonuclease I assay, T7E1 (indicated as % indels; gel not shown) are reported below the gel. PO, phosphodiester; PS, phosphorothioate. Fifty-base-pair DNA ladder (NEB) was shown. See also Figure S1 for integration activity of dsODNs.

(B) ssODNs, but not dsODNs, direct precise sequence insertions. The frequency of the different types of indels, evaluated by deep sequencing, are reported: % indels w/o KI (gray) and KI events that include the precise KI events (black) and KI with additional indels (hatched).

(C) Influence of location and nature of the modification in the oligonucleotide on genome editing efficiency. Different parameters were evaluated: the type of chemical modification (LNA [locked nucleic acid]), the number of modified nucleotides (two at both ends or at 30 end in ss2sPS or ss2sPS_30 , resp.; three at both ends or at the 30 end in ss2s-3PS or ss2s-3PS_30 and ss2s-3LNA_30 , resp.). RFLP rate is shown for different modified oligonucleotides, as mean ± SD (3–20 independent experiments). For all the modified ssODNs compared to the PO ODN, p values from Mann-Whitney statistical analysis are <0.05.

Optimization of Short ODN Donor Design(JB Renaud, et al.,2016)