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[Preprint]. 2023 Mar 23:2023.03.20.533459. [Version 2] doi: 10.1101/2023.03.20.533459

Figure 2. Protecting oligonucleotide (P.O.) enhances the potency and stability of C20 crRNA.

Figure 2.

a). Schematic representation of the P.O. strategy. Initial annealing of the anti-repeat region of the tracrRNA to the repeat region of the crRNA is hypothesized to lead to P.O. displacement via duplex invasion. b). Schematic representation of the design and modifications of different lengths of P.O.s. c-f). Dose-response curves of C20 targeting different sequences with and without P.O.s in HEK293T-SpyCas9-tracrRNA-TLR-MCV1 reporter (c), HEK293T-SpyCas9-ABE-tracrRNA-dGFP reporter (d), and Hepa1–6-SpyCas9-ABE-tracrRNA stable cell lines (e-f). Editing efficiencies were quantified by FACS (c-d) or targeted amplicon deep sequencing (e-f) (n = 3 biological replicates). Data represent mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (two-way ANOVA; C20 was used as control in multiple comparisons). g). The stability of C20 annealed with different lengths of P.O.s (see b) in 10% non-heat-inactivated FBS at 37°C for the indicated numbers of hours. Oligos were resolved by 10% urea-denaturing PAGE followed by SYBR Gold staining.