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CRISPR
 

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. 

Figure 1. Different steps of DNA targeting by CRISPR-RNA

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.

Figure 2. smFRET assay to study DNA interrogation i.e.(recognition-rejection) by Cas9-RNA.
(a) Schematic of single-molecule FRET assay to study DNA interrogation by Cas9-RNA. Specific interaction between Donor (Cy3) labeled and surface immobilized DNA target and acceptor (Cy5) labeled Cas9-RNA in solution results in FRET, where different FRET values report also report on nature of these interactions. (b) DNA targets with different positions and extent of off-target bp (mismatches) were used in these experiments. (c) A representative smFRET time-trajectory showing the real-time interaction between Cas9-RNA and a DNA target molecule with 12 PAM-distal mismatches. DNA targets with < 12 PAM-distal mismatches were ultra-stably bound by Cas9-RNA. (d) These experiments with different DNA targets helped us uncover bimodal binding nature of Cas9-RNA i.e. Cas9-RNA binds DNA in predominantly two distint sub-steps, first being a PAM-surveillance step and if PAM is detected then the DNA unwinding ensues leading to second sub-step.
Figures adapted from : Nat. Commun. 7:12778 doi: 10.1038/ncomms12778 (2016).

  Figure 3. Irreversible and reversible binding by Cas9-RNA.  Irreversible (ultra-stable) DNA binding by Cas9-RNA for any DNA with ≥ 9 PAM-proximal matching bp (left). Binding becomes reversible (right) only with &lt; 9 PAM-proximal matching bp. High tolerance to PAM-distal mismatches and high sensititivty to PAM-proximal mismatches for stable binding further supports the model of unidirectional of DNA unwinding from PAM-proximal to PAM-distal end, as shown above. Internal dynamics of DNA unwinding and rewinding of a Cas9-RNA-DNA is not shown in these schematics. They were investigated in another project and have been discussed below.

Figure 3. Irreversible and reversible binding by Cas9-RNA.
Irreversible (ultra-stable) DNA binding by Cas9-RNA for any DNA with ≥ 9 PAM-proximal matching bp (left). Binding becomes reversible (right) only with < 9 PAM-proximal matching bp. High tolerance to PAM-distal mismatches and high sensititivty to PAM-proximal mismatches for stable binding further supports the model of unidirectional of DNA unwinding from PAM-proximal to PAM-distal end, as shown above. Internal dynamics of DNA unwinding and rewinding of a Cas9-RNA-DNA is not shown in these schematics. They were investigated in another project and have been discussed below.

My 3 minute case for doing smFRET for CRISPR that I presented to a general non-specialist audience.