Fig. 6.
Improved binding affinity of the crRNA-AsCpf1 complex by the U-rich 3′-overhang. a The endogenous levels of the crRNAs were assessed by northern blot analysis following transfection of the crRNA template PCR amplicons into HEK293-T cells. The intracellular levels of 38s/18s RNA were used as a loading and quality control of the endogenous RNAs. The transfection efficiency was also controlled by the PCR intensities of the crRNA template DNA isolated from the transfected HEK293-T cells. This result is representative of three repeated experiments. b The indel efficiency was monitored at a DNMT1 locus following transfection of the AsCpf1 plasmid constructs and synthetic guide RNAs into HEK293-T cells. The 3′-terminal four nucleotides of crRNA and sgRNA for AsCpf1 and SpCas9, respectively, were covalently linked with the phosphorothiate group. *p = 0.008, **p = 0.39 (n = 3), two-tailed Student’s t test. c The indel efficiency was measured using the crRNA and tracrRNA of the CRISPR-Cas9 system. The truncated crRNAs with a length of 53 nt and 63 nt were compared with those with the U4AU4 3′-overhang. This gel image is representative of three repeated experiments. d Binding experiments were conducted using a microscale thermophoresis (MST) system. The binding affinities of guide RNAs and the effector proteins (SpCas9 and AsCpf1) were measured using Monolith NT.115 (NanoTemper Technologies GmbH). All samples were loaded into NanoTemper standard capillaries and repeated three times for each measurement. e Isothermal titration calorimetry (ITC) measurements were conducted on an Auto-iTC200 Microcalorimeter (GE Healthcare) at 25 °C in PBS buffer (pH 7.4). The purified recombinant AsCpf1 proteins (5 μM) were titrated with 50 μM synthetic crRNAs with 2 μL injections. Ka = (1.90±0.87) × 108 M−1 for the U-rich crRNA and (1.15±0.54) × 107 M−1 for the canonical crRNA. ΔH = –31.92±1.79 kcal/mol and −22.86±1.86 kcal/mol for the U-rich and canonical crRNA, respectively. The values are the average of three independent experiments