Unmodificated stepless regulation of CRISPR/Cas12a multi-performance

Abstract

As CRISPR technology is promoted to more fine-divided molecular biology applications, its inherent performance finds it increasingly difficult to cope with diverse needs in these different fields, and how to more accurately control the performance has become a key issue to develop CRISPR technology to a new stage. Herein, we propose a CRISPR/Cas12a regulation strategy based on the powerful programmability of nucleic acid nanotechnology. Unlike previous difficult and rigid regulation of core components Cas nuclease and crRNA, only a simple switch of different external RNA accessories is required to change the reaction kinetics or thermodynamics, thereby finely and almost steplessly regulating multi-performance of CRISPR/Cas12a including activity, speed, specificity, compatibility, programmability and sensitivity. In particular, the significantly improved specificity is expected to mark advance the accuracy of molecular detection and the safety of gene editing. In addition, this strategy was applied to regulate the delayed activation of Cas12a, overcoming the compatibility problem of the one-pot assay without any physical separation or external stimulation, and demonstrating great potential for fine-grained control of CRISPR. This simple but powerful CRISPR regulation strategy without any component modification has pioneering flexibility and versatility, and will unlock the potential for deeper applications of CRISPR technology in many finely divided fields.

Graphical Abstract

Scheme 1.

(A) Modification of the recognition component crRNA or the execution component Cas nuclease is difficult and rigid, and allows achievement of only rough regulation of some CRISPR/Cas performances. (B) Adding the RNA complementary strands as accessories to the original components is simple and flexible, and allows precise control of many CRISPR/Cas performances by selecting different ERAs to alter the dynamics and thermodynamics of activator binding. ‘ON/OFF’ indicates that Cas12a is in ‘activatable/inactivatable’ state.

Figure 1.

(A) ERAs toolkit bind to crRNAs to form double-stranded RNA substrates with toeholds of different directions and lengths, and activators activate Cas12a via TMSD. Activation efficiency of ERA–crRNA–Cas12a with different lengths of 5′ toehold (B) or 3′ toehold (C). (D) Relationship between toehold length and direction on the ERA–crRNA–Cas12a catalytic efficiency (k_cat/K_M). (E) Mismatched ERAs toolkit bind to crRNA to form substable substrates, and eliminating the unstable state can accelerate ERA-controlled Cas12a activation. Activation efficiency of ERA–crRNA–Cas12a (different ERA mismatch sites) with 3′ toehold-6 nt (F) or −7 nt (G), 5′ toehold-4 nt (H) or −7 nt (I). ERA (−) means no ERA is added. Orange represents the toehold in the crRNA. Error bars represented the standard deviation calculated from three independent experiments.

Figure 2.

(A) Discrimination of single-base-mismatched activators by uncontrolled or ERA-controlled Cas12a. (B) DF of ERA-controlled Cas12a with different 5'-toehold lengths in the recognition of different mismatched activators. (C) Activation efficiency of ERA-controlled Cas12a by different mismatched activators when toehold direction is 5' or 3' and length is 7 nt or 9 nt. (D, E) Relationship between discriminantion factors and mismatch site, toehold length and direction. Orange represents the toehold in the crRNA. ERA (-) means no ERA is added. Error bars represented the standard deviation calculated from three independent experiments.

Figure 3.

(A) When specificity is dominated by kinetics/TMSD, ERA-controlled Cas12a can hardly discriminate mismatches far from the toehold. (B) When specificity is dominated by thermodynamics/TE, ERA-controlled Cas12a discrimination is independent of mismatch position. The fluorescence kinetics (C, D, E, F) and discrimination factors (G, H, I, J) of TE-based ERA-controlled Cas12a identifying different mismatched activators. Orange represents the forward toehold exposed before TE reaction, and green represents the reverse toehold exposed after TE reaction. ERA (−) means no ERA is added. Error bars represented the standard deviation calculated from three independent experiments.

Figure 4.

(A) In one-pot assay, the CRISPR/Cas system and the amplification system are spatially and temporally continuous, so the negative feedback effect leads to low detection efficiency. (B) The timing-activated ERA-controlled Cas12a system and the amplification system are spatially continuous but temporally isolated, independent of negative feedback effects to achieve high detection efficiency. The signal output efficiency of PER and CRISPR’s one-pot or two-step assay without (C) or with (D) ERA support. The sensitivity of one-step assays without (E) or with (F) ERA support. The signal-to-noise ratio of the one-pot or two-step assay without (G) or with (H) ERA support. Orange represents the toehold in the crRNA. ERA (−) means no ERA is added. Error bars represented the standard deviation calculated from three independent experiments.