CRISPR creates site-specific double-strand breaks (DSBs) in living cells. The system requires two components: a guide RNA (gRNA) to target a specific DNA sequence, and a CRISPR-associated endonuclease (Cas9 protein) to perform the actual cleavage. While the actual cleavage is quick, the initial steps take many hours: transfection of the cells, expression of the guide RNA and/or Cas9 protein, nuclear localization, target searching, and eventual formation of the gRNA-DNA-protein complex at the correct site. Because of this, the actual cleavage events are also spread out over a long period of time. Traditional CRISPR-Cas9 editing can also be inaccurate, with many DSBs happening off-target at locations far away from the intended site.

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How CRISPR becomes Very Fast: A 365 nm wavelength stoplight

The Johns Hopkins team, led by Profs. Taekjip Ha and Bin Wu created vfCRISPR by photocaging specific deoxythymidine residues of the guide RNA resulting in a caged guide RNA (cgRNA). This cage still allows the cgRNA and endonuclease to bind at the target DNA sequence, but the cages physically block the HNH portion of Cas9 from cleaving DNA. Illumination with 365 or 405 nm light releases thymidine from its cage, allowing DNA cleavage to proceed. To synchronize cleavage across an entire cell sample, the CRISPR components are first allowed to incubate and complete the slow localization and binding steps of the process. This ensures the cgRNA-DNA-protein complexes have already formed and are primed to begin cleavage before the ~30 second illumination period begins.

Benefits of vfCRISPR

  • Fast cleavage: Normally DNA cleavage by CRISPR-Cas9 is spread over many hours, as it takes time for the gRNA and Cas9 protein to enter the nucleus and bind to the correct DNA sequence. With vfCRISPR, 50% of the target sequences can be cleaved within 30 seconds of illumination.
  • Fast Repair. Ensuing DNA damage repair was faster and more concerted compared to repair after other methods of DNA damage. The team found that DNA damage response (DDR) proteins were recruited within 2 minutes of cleavage and repair was complete within 15 minutes.
  • Increased accuracy and precision. Both accuracy and precision of cleavage were improved, with fewer off-target strand breaks and shorter deletions of more consistent length. It was also possible to target just a single allele for cleavage. Allele-specific CRISPR is being used to create isogenic iPSC cell lines for both disease models and also for potential cell therapies.
  • Few artifacts. No observed phototoxic effects were observed in the cells after illumination.

The combination of control in both time and location makes vfCRISPR a likely tool for studying DNA damage repair. Frequently these studies have been performed using much less precise methods of damaging DNA, resulting in more variability in the observed kinetics of the ensuing DNA damage response. But with vfCRISPR, all of the damage is to the same gene at the same time, so the kinetics and position dependence of DNA repair can be studied more accurately.

The photochemistry:

vfCRISPR uses 6-nitropiperonyloxymethyl caged deoxythymidine (NPOM-caged-dT) to arrest cleavage. Several caged-dT residues are added to the distal end of each guide RNA. Located there they don’t interfere with binding to the targeted DNA sequence or binding of Cas9 to the DNA-RNA complex but do prevent DNA cleavage. Exposure of the caged dT residues to UV light begins a two-step process which results in the generation of the uncaged thymidine, allowing cleavage to proceed. The Johns Hopkins team found that 1.3 J/cm2 (~ 40 mW/cm2 for 30 seconds) of 365 nm light was sufficient to uncage cgRNAs.

NPOM-caged-dT modified RNA as well as phosphoramidites are available from Glen Research and Gene Link.

Liu, Y., Zou, R. S., He, S., Nihongaki, Y., Li, X., Razavi, S., … & Ha, T. (2020). Very fast CRISPR on demand. Science, 368(6496), 1265-1269.