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In microbes, clustered regularly interspaced short palindromic repeats (CRISPR) systems provide adaptive immunity against foreign DNA by utilizing CRISPR nuclease in complex with a RNA (CRISPR-RNA) to target foreign DNA for their cleavage impairment. CRISPR-RNA targets DNA with sequences complementary to RNA with a requirement that they be adjacent to a special motif called protospacer adjacent motif (PAM). Upon binding, CRISPR-RNA unwinds dsDNA target and the RNA of CRISPR-RNA forms a heteroduplex with complementary strand of the unwound DNA target resulting in a three-stranded nucleic acid structure, also known as R-loop.
These nucleases, which can be programmed via RNA, to bind and cleave any DNA has been repurposed as revolutionary genome engineering tools, where both their DNA binding and DNA cleavage functions are being used for wide-ranging applications including gene editing, regulation, visualization. But resolving CRISPR-RNA’s off-target activity problems along with a better understanding of its molecular mechanisms are near prerequisites for its efficient and accurate use, rational engineering and more widespread adoption especially for clinical applications.
Single molecule imaging for molecular mechanism of CRISPR
There are many steps in DNA targeting by CRISPR-RNA, some of which are summarized in Figure 1. Understanding of CRISPR's molecular mechanism will improve with individually and combinatorially investigating these steps which has been my goal during my PhD. I have employed single molecule imaging (chiefly smFRET), complemented with biochemical assays, to investigate these steps. Single molecule techniques for ideal for such investigations because:
observe wide ranging events (transient to long-lived).
detect rare events.
identify distinct sub-steps (for e.g. distinct FRET values for distinct sub-steps)
evaluate intrinsic heterogeneity of a biological system by identifying molecular sub-populations
all the above in real-time which allows for a kinetic analysis of distinct sub-steps.
I have performed these relevant investigations with different DNA sequences i.e. with and without off targets. So, the changing nature of a given step with off-targets not only helps in aiding the molecular understanding of it but also its specificity. Judicious selection of fluorophore labeling positions can allow you to investigate any of the given steps either individually or combinatorially. One of many such labeling geometries is shown below (Figure 2), which we employed to investigate how Cas9-RNA recognizes/rejects different DNA sequences.
We have also investigated other important steps of CRISPR-RNA targeting using a similar approach and extended it to CRISPR-Cpf1 family and other CRISPR-Cas9 variants which are currently the most widely used genome engineering CRISPR tools. The obtained information can help us understand the mechanism of these genome engineering tools, design strategies to improve their efficiency and accuracy, perform rationale-guided engineering of new CRISPR tools and CRISPR inhibitors/activators.