Fig. 4: Rational design of guide CBS in STAR.

a Schematic of working principle of STAR for NRAS transcript degradation in HEK293T cells. b Diagram showing the design of dEcCas6-barnase fusion coupled with the ssRNA-binding protein ORF5. c Evaluation of knockdown efficiency of dEcCas6-barnase 17 and dEcCas6-barnase 11 with or without the ORF5 at the N terminus. bgCBS utilized either a random 40-nt spacer sequence (referred to as non-targeting) or a 40-nt targeting spacer sequence. d Analysis of NRAS-targeting bgCBS abundances in HEK293T cells transfected with either dEcCas6-barnase 17 or dEcCas6-barnase 11, with or without ORF5. Data are normalized to the group transfected with NRAS-targeting bgCBS and dEcCas6-barnase 17. e Evaluation of NRAS transcript knockdown efficiency using mgCBS constructs with wtCBS at the 5ʹ end (5ʹ mgCBS) or the 3ʹ end (3ʹ mgCBS), and bgCBS containing wtCBS. f Knockdown efficiency of NRAS transcript using bgCBS constructs incorporating enCBS variants with varying affinities to dEcCas6. bgCBS constructs were prepared using enCBS (m1-5), enCBS (t-2), enCBS (loop10), and enCBS (d22-29), with results normalized to the non-targeting bgCBS. Heterozygous bgCBS constructs were prepared with enCBS (d22-29) at the 5ʹ end (5ʹ d22-29) or at the 3ʹ end (3ʹ d22-29), and wtCBS at the opposite end. The black arrows indicate the 8-nt 3ʹ flanking. The red lines represent the spacers in bgCBS constructs. Data are mean ± s.e.m. of n = 3 biological replicates. Source data are provided as a Source data file.