tCº, a tricyclic fluorescent cytosine analog enables the study of CRISPR kinetics and structures

tC^O, a tricyclic fluorescent cytosine analog enables the study of CRISPR kinetics and structures

1,3-Diaza-2-oxophenoxazine, commonly referred to as tC^O, is a tricyclic fluorescent cytosine analog used in CRISPR kinetic and structural studies as an internal, non-disruptive fluorescent probe. tC^O shares structural homology with natural cytosine and forms highly stable base pairs with guanine, allowing incorporation into single-guide RNA (sgRNA) or target DNA without altering native conformation or disrupting Cas9 activity. tC⁰ is one of the brightest intrinsically fluorescent nucleoside analogs reported in duplexes, brighter than alternatives like 2-aminopurine, and it maintains fluorescence even when surrounded by guanine residues.

Researchers use tC⁰ in fluorescence resonance energy transfer (FRET) studies and to monitor nucleic acid dynamics, hybridization, and transcription in real time, such as tracking mRNA translation in living cells. tC⁰ can serve as a core structural element for modified nucleobases such as the G-clamp, which enhances antisense oligonucleotide (ASO) binding affinity and target knockdown efficacy, for example, by targeting long non-coding RNAs in muscle tissues.

Liu et al. (2020) reported that engineered Cas9 variants improve DNA target discrimination by slowing the catalytic cleavage step, allowing mismatched (off‑target) DNA time to dissociate before strand cutting occurs. The research group performed kinetic analyses of the high-fidelity (Cas9-HF1) and hyper-accurate (HypaCas9) engineered Cas9 variants and showed that DNA cleavage is impaired by more than 100-fold in the high-fidelity variants. The high-fidelity variants improve discrimination by slowing the observed cleavage rate without increasing the rate of DNA rewinding and release, favoring release over cleavage of a bound off-target substrate because the cleavage rate is low.

Cas9 uses a guide RNA to bind DNA and check for complementarity. Once bound, it undergoes conformational changes that activate its nuclease domains and cleave both DNA strands. Binding is fast, even with partial mismatches. In non-engineered CRISPR/Cas9, cleavage is also fast; therefore, off‑target sites can be cut before the complex dissociates.

A kinetic profile is essential for off‑target editing; Liu et al.'s engineering strategy aimed to slow cleavage to improve fidelity. Several engineered Cas9 variants modify residues in the HNH nuclease domain or the REC lobe, which control the conformational checkpoint before cleavage, to slow HNH activation and DNA cleavage while keeping DNA-binding kinetics mostly unchanged, allowing mismatched DNA to dissociate before cleavage occurs.

Liu et al. used a stopped-flow instrument to measure R-loop formation rates for all enzymes by monitoring tCo fluorescence at −16 nt. This tricyclic cytosine analog is quenched by base stacking in dsDNA. Opening the duplex causes a large increase in fluorescence.

In the stopped-flow experiment, the researchers mixed the Cas9-gRNA complex (1:1 ratio) with a tCo-labeled 55/55 nt DNA substrate at 37°C using an AutoSF-120 stopped-flow instrument (KinTek Corporation, Austin, TX). To monitor the reaction, the research group excited the fluorophore at 367 nm and monitored the emission at 445 nm using a single band-pass filter with a 20 nm bandwidth. However, for the Cy3/Cy5 FRET signal, the researchers excited samples at 550 nm and monitored the time-dependent fluorescence change at 670 nm using a single band-pass filter with a 30-nm bandwidth (Semrock).

The study found that

·         DNA unwinding occurs more rapidly near PAM and is largely unchanged in Cas9 variants.

·         Cas9 specificity is governed by kinetic partitioning.

·         HypaCas9 and Cas9-HF1 show slow observed rates of cleavage.

·         Engineered Cas9s improve specificity by decreasing the rate of DNA cleavage.

·         Kinetic partitioning governs Cas9 specificity.

As a result, Cas9 is shifted from a "bind‑and‑cut" enzyme to a "bind‑check‑wait‑cut" enzyme. Slower cleavage increases specificity because off‑target sites typically bind more weakly due to mismatches. When cleavage is fast, even weakly bound DNA may be cut. When cleavage is slow, weakly bound DNA dissociates before the nuclease domains activate. On‑target DNA remains bound long enough for cleavage. In this kinetic proofreading mechanism, discrimination arises from timing, not just affinity.

The engineered CRISPR/Cas9 reaction pathway discriminates between on-target and off-target DNA by slowing cleavage to allow off-target DNA to dissociate. This slower reaction starts in a DNA-competent state and proceeds through DNA binding and R-loop completion to the final cleavage state.

References

Berry, D. A.; Jung, K.; Wise, D. S.; Sercel, A. D.; Pearson, W. H.; Mackie, H.; Randolf, J. B.; Somers, R. L. Tetrahedron Lett 2004, 45, 2457-2461. Tetrahedron Letters

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Engman KC, Sandin P, Osborne S, Brown T, Billeter M, Lincoln P, Nordén B, Albinsson B, Wilhelmsson LM. DNA adopts normal B-form upon incorporation of highly fluorescent DNA base analogue tC: NMR structure and UV-Vis spectroscopy characterization. Nucleic Acids Res. 2004 Sep 27;32(17):5087-95. [PMC]

Füchtbauer AF, Preus S, Börjesson K, McPhee SA, Lilley DMJ, Wilhelmsson LM. Fluorescent RNA cytosine analogue - an internal probe for detailed structure and dynamics investigations. Sci Rep. 2017 May 24;7(1):2393. [PMC]

G-clamp-improves-the-thermodynamic-stability-of-the-DNA-duplex

Liu MS, Gong S, Yu HH, Jung K, Johnson KA, Taylor DW. Engineered CRISPR/Cas9 enzymes improve discrimination by slowing DNA cleavage to allow release of off-target DNA. Nat Commun. 2020 Jul 17;11(1):3576. [PMC]

Sandin P, Wilhelmsson LM, Lincoln P, Powers VE, Brown T, Albinsson B. Fluorescent properties of DNA base analogue tC upon incorporation into DNA--negligible influence of neighbouring bases on fluorescence quantum yield. Nucleic Acids Res. 2005 Sep 7;33(16):5019-25. [PMC]

Sandin P, Börjesson K, Li H, Mårtensson J, Brown T, Wilhelmsson LM, Albinsson B. Characterization and use of an unprecedentedly bright and structurally non-perturbing fluorescent DNA base analogue. Nucleic Acids Res. 2008 Jan;36(1):157-67. [PMC]

Sandin P, Stengel G, Ljungdahl T, Börjesson K, Macao B, Wilhelmsson LM. Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment. Nucleic Acids Res. 2009 Jul;37(12):3924-33. [PMC]

The Glen Report, Glenresearch.

The Glen Report, Glen Report 22.13 - tC,