Figure 2.
Chemical modifications of guide sequences. (a) crRNAs with 2′OMe modifications and without modifications (native strand). The pink highlighted nucleotides in the guide sequences that interact with Cas9 protein at the 2′ OH group. (b–d) crRNA with various patterns of modifications using 2′OMe (b) or 2′F (c) or PS (d). (e) Combination of structure-guided (SG) chemical modification of 2′OMe or 2′F with PS in the guide sequences. HEK293 cells described in Figure 1a were incubated with crRNA in (b–e) and an unmodified tracrRNA. FACS was performed to determine the ratio of GFP− cells. (f) crRNAs targeting HBB and EMX-1 were chemically modified with patterns described in e (5′-PS-2′OMe_SG-PS-2′F), and TIDE analysis was performed to determine indels at the HBB and EMX-1 loci, respectively. (g) crRNAs targeting GFP with modifications of single or multiple 2′OH in the guide region. TIDE analysis was performed to measure indels at GFP locus. *P < 0.05. (n = 4 in f and g, and n = 3 in others), error bars as s.d.