Figure 2.

Modified chimeric guide RNA backbone enhances editing efficiency in HSPCs. CD34⁺ HSPCs were transduced with lentivirus expressing sgRNAs encoding chimeric guide RNA backbone 1 (chRNA1) or 2 (chRNA2) on day 1, followed by electroporation of Cas9 mRNA or protein on day 4. Editing was then assessed by flow cytometry 4 days post-electroporation on day 8. (a) Overview of structural changes between the standard chimeric guide RNA backbone 1 (chRNA1) versus the modified chimeric guide RNA backbone 2 (chRNA2). This figure is modified from Chen et al.⁸ (b) Overview of optimized experimental outline for comparing chimeric guide RNA backbone configurations. (c) Representative FACS plots of CD45 editing. (d) CD45 editing efficiency assessed by flow cytometry. Statistical significance was calculated using unpaired student’s t-test. **p = 0.0058. (e) Representative FACS plots of CD44 editing. (f) CD44 editing efficiency. (g) Representative FACS plots of CD29 editing. (h) CD29 editing efficiency. (i) Comparison of editing efficiency between chRNA1 and chRNA2 in terms of % negative cells, for different sgRNAs with different gene targets (CD45, CD44, CD29), measured by flow cytometry 4 days post-electroporation. Statistical significance was calculated using paired student’s t-test. **p = 0.0031. Error bars represent standard deviation (s.d.).