Fig 1.
Unmodified crRNA:tracrRNA require a sequential electroporation method with Cas9 mRNA for gene editing.
Sequential electroporation involves electroporation of Cas9 mRNA, followed 6 hours later by electroporation of synthetic guide RNA and harvested 2–3 days later for analysis. With a co-electroporation method, both Cas9 mRNA and synthetic guide RNA are delivered into cells at the same time, then harvested 2–3 days later. No detectable (n.d.) gene editing was observed with unmodified synthetic crRNA:tracrRNA in co-electroporation (Co) with Cas9 mRNA into K-562 cells for three gene targets, but resulted in a significant increase when with sequential electroporation (Seq, duplicate samples). UT = Untreated, NTC = Non-targeting control, M = DNA ladder.
Fig 2.
Minimal modification of guide RNA improves stability to increase gene editing efficiency with Cas9 mRNA in co-electroporation.
A. Modification patterns of synthetic sgRNA (100-mer), crRNA (42-mer) and tracrRNA (74-mer) with one to three 2’-O-methyl modifications with 3’ phosphorothioate linkages (MS, denoted with red *). 1x-3xMS modifications were added to both ends of crRNA, tracrRNA and sgRNA (left column) or the 5’ or 3’ end of crRNA and tracrRNA (right column) and tested in the different combinations shown. Unmodified and modified synthetic guide RNAs targeting genes PPIB, PSMD7 and PSMD11 were co-electroporated with Cas9 mRNA into K-562 cells (duplicate samples shown; B-D). B. Co-electroporation of Cas9 mRNA and 1x-3xMS-modified synthetic sgRNA all showed similar levels of gene editing as estimated from a DNA mismatch detection assay. C. Unmodified (unmod) crRNA:tracrRNA produced no detectable (n.d.) editing for any of the gene targets, while 1x-3xMS-modified crRNA and tracrRNA had detectable, but varying, levels of gene editing efficiencies. D. Single-end modifications on crRNA and tracrRNA indicated that modification of the 5’ end of crRNA is important for stability for efficient gene editing. The numbers under each gel image are percentage of gene editing. UT = Untreated; NTC = Non-targeting control, M = DNA ladder.
Fig 3.
Addition of MS modifications on guide RNAs may improve gene editing efficiency with Cas9 protein in co-electroporation of RNPs.
Cas9 protein and unmodified or modified synthetic guide RNAs targeting PPIB, PSMD7 and PSMD11 were complexed and delivered as RNPs into K-562 cells using co-electroporation (duplicate samples shown). A. For most modification patterns, similar gene editing efficiencies were detected with both ends modified sgRNA when compared to unmodified (unmod) sgRNA; only one gene (PSMD7) showed increased (1.8-fold) gene editing with 1x-2xMS modifications. B. Co-electroporation of both ends and single-end modified crRNA:tracrRNA for genes PSMD7 and PSMD11 with Cas9 protein did not result in consistent increase or decrease in gene editing efficiencies compared to unmodified, but increased gene editing with some modification patterns targeting PPIB. The numbers under each gel image are percentage of gene editing. UT = Untreated, NTC = Non-targeting control, M = DNA ladder.
Fig 4.
MS modifications of guide RNA produce similar gene editing efficiency to unmodified when lipid transfected into a stably expressing Cas9 cell line and some modification patterns are toxic to cells.
A. Gene editing efficiency of unmodified (unmod) and modified crRNA:tracrRNA or sgRNA showed similar levels of gene editing efficiencies (< 1.5-fold difference) for each gene when transfected into a stably expressing Cas9 U2OS cell line. Error bars are representative of biological triplicates. B. Average cell viability of unmodified or modified guide RNAs for all three genes resulted in a significant decrease in cell viability (< 60%) for some modification patterns (gray bars). NTC = Non-targeting control. Error bars are representative of the average of all three genes with the same modification pattern over two (PPIB) or three (PSMD7 and PSMD11) independent experiments.
Fig 5.
Modest improvement of gene editing efficiency with some MS-modified gRNAs was observed using Cas9 mRNA and Cas9 protein in lipid co-transfections.
Unmodified (unmod) and modified guide RNAs were co-transfected with Cas9 mRNA (A and B) or Cas9 protein (C and D). Gene editing efficiencies were estimated for PPIB-targeting guide RNAs in U2OS cells (light blue bars) and HeLa cells (dark blue bars; A and C). Functional gene knockout was quantified using a phenotypic analysis with PSMD7- (light blue bars) or PSMD11- (dark blue bars) targeting guide RNAs in a Ubi-EGFP U2OS cell line (B and D). Cell viability was assessed for each lipid transfection experiment: U2OS cells (dark green boxes) and HeLa cells (light green boxes; A and C) and PSMD7 (dark green boxes) and PSMD11 (light green boxes; B and D). NTC = Non-targeting control. Error bars representative of biological triplicates.
Table 1.
Summary of results from modifying gRNAs in different applications compared to unmodified gRNAs.
A comparison of unmodified and modified gRNAs in different applications, from electroporations to lipid co-transfections, based on the results in this paper. N.d. = not determined, * some modification patterns can be toxic to cells.