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. 2022 Apr 18;62(24):3512–3520. doi: 10.1021/acs.biochem.1c00768

Figure 1.

Figure 1

Incorporation of 2′-O-methyl-3′-PACE (MP) modifications at the 3′ end of chemically synthesized sgRNAs promotes stability and editing yields. (A) Structures of chemical modifications used in synthetic gRNAs and schematic structures of two examples of sgRNAs with MS or MP 3′ end modifications. (B) sgRNAs modified by 3xMS at the 5′ end and various modification schemes at the 3′ end (as indicated) were transfected into K562 cells in the absence of Cas protein. The amounts of the various gRNAs present at different time points following transfection were measured by qRT-PCR. (C and D) sgRNAs modified by 3xMS at the 5′ end and various modification schemes at the 3′ end, as indicated, without or with an additional MP modification at position 5 to improve specificity, were cotransfected with Cas9 mRNA in HepG2 cells (C) as well as in primary human T cells (D). (E) K562 cells were cotransfected with BE4-Gam mRNA and synthetic sgRNA modified by 3xMS or 3xMP at the 3′ end. Indel and editing yields were measured by deep sequencing of PCR amplicons of the HBB target locus (HBB_ON) and a known highly reactive off-target site on chromosome 9 (HBB_OFF1). Bars on bar graphs or dots and error bars on line graphs represent means with standard deviation (n = 3).