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'Very Fast' CRISPR Enables Immediate, Synchronous Cleavage of Genomic Sites

NEW YORK – Researchers at Johns Hopkins University have developed a gene editing approach they call very fast CRISPR (vfCRISPR) that is capable of creating double-strand breaks (DSBs) in seconds, and at the submicrometer scale.

During gene editing, it can take hours for a guide RNA (gRNA) to guide the Cas9 nuclease to the correct DNA sequence. To better control Cas9's action, the researchers caged the gRNA with light-sensitive nucleotides, so that it could bind Cas9 to its DNA target but keep the enzyme from cutting until the gRNA was exposed to light.

As they described in a study published on Thursday in Science, the investigators found that the synchronized cleavage improved kinetic analysis of DNA repair, revealing that cells responded to Cas9-induced DSBs within minutes. Further, they used single-cell fluorescence imaging to characterize multiple cycles of the formation and dissolution of 53BP1 repair foci.

"Imaging-guided subcellular Cas9 activation further facilitated genomic manipulation with single-allele resolution," the authors wrote. "vfCRISPR enables DNA-repair studies at high resolution in space, time, and genomic coordinates."

The design principle of vfCRISPR was based on the Streptococcus pyogenes Cas9 cleavage mechanism. On the basis of the researchers' mechanistic understanding of the nuclease's protospacer adjacent motif (PAM), they replaced two or three uracils at the PAM-distal region of the crRNA with light-sensitive, 6-nitropiperonyloxymethyl-modified deoxynucleotide thymine caged nucleotides, forming a caged gRNA (cgRNA) when hybridized to wild-type transactivating CRISPR RNA (tracrRNA). The Cas9/cgRNA complex retained the ability to bind its target DNA but couldn't cleave because the steric hindrance imposed by the caging groups prevented full DNA unwinding and nuclease activation.

Once the caging groups were removed on exposure to light stimulation, however, the now-activated Cas9/cgRNA complex cleaved the target DNA within seconds in vitro. The researchers also characterized the activity of vfCRISPR in HEK293 cells by targeting four endogenous loci, and found light-induced indel efficiency of up to 97 percent, whereas cells without light exposure had almost no detectable indels.

"Compared with other Cas9 induction methods, vfCRISPR exhibited much faster cleavage kinetics and higher cleavage efficiency," the authors wrote. "We attribute the very fast kinetics to skipped nuclear localization or target-searching steps, and the higher cleavage efficiency to the use of wild-type Cas9."

When they conducted a genome-wide analysis of off-target editing, the investigators also found reduced off-target activity from vfCRISPR compared with wild-type gRNA, consistent with improved specificity from deoxyribonucleotide incorporation into the gRNA and demonstrating that cgRNA enables very fast and efficient inducible DNA cleavage in mammalian cells. Importantly, the researchers said, having a precisely defined time for cleavage allows for the investigation of the generation and repair kinetics of Cas9-mediated DSBs.

"To the best of our knowledge, vfCRISPR provides the highest spatial and temporal resolutions to induce site-specific DSBs in living cells. This study sets the blueprint for further systematic studies of the DDR that combine vfCRISPR with time-resolved biochemical, sequencing, and imaging readouts," the authors concluded. "The use of cgRNA with other Cas9-based systems such as nickases, base editors, and prime editors may facilitate the study of single-strand break, base excision or mismatch, and flap repair, respectively."

Further, they noted, combining vfCRISPR with subcellular photoactivation potentially enables precise genome editing with single-allele specificity and elimination of off-target activity.

In a companion Perspectives column published in Science on Thursday, Memorial Sloan Kettering Cancer Center molecular biologist Maria Jasin and NIH Fellow Darpan Medhi wrote that the vfCRISPR approach hones CRISPR-Cas9 from the blunt instrument it is today into "a precision instrument that is both temporally and spatially controlled."

Until now, cleavage of genomic sites has not been immediate and hasn't usually been synchronous, hampering the study of DNA breakage and repair in real time, but vfCRISPR overcomes these limitations. Using this technique will make it possible to interrogate a cell's response to DSBs in real time, allowing for greater understanding of how cells maintain genomic integrity in the face of such lesions, Jasin and Medhi noted.

They also highlighted specific findings from the study. For example, one experiment that modeled repair kinetics at one target site unexpectedly suggested repetitive cleavage and repair, though this was possibly the result of the site being particularly vulnerable to one type of repair product.

"With time-resolved chromatin immunoprecipitation, the recruitment and retention of components of the DNA damage signaling and repair machinery could be tracked within minutes, including the rapid responder MRE11," Jasin and Medhi wrote. "Spreading of a commonly used marker of DSBs, phosphorylated histone H2AX, could be resolved into two rates, one estimated at about 150 kb/min and a surprising second layer at about 460 kb/min, reaching an astounding 30 Mb from the DSB within 1 hour. Other methods to study DNA repair are less controllable."

Presumably, Medhi and Jasin added, the focused application of light could also prevent off-target activity. And though it remains to be seen how well the approach of light activation through cgRNAs translates to studying other processes, it holds promise for the precise interrogation of other DNA repair pathways.