XNA

Idea:

The goal of this project is to determine the compatibility of the CRISPR/Cas9 genome editing system with various xenonucleic acids (XNAs). XNA is a synthetic nucleic acid similar to DNA or RNA, but with the ribose or deoxyribose sugar replaced with another molecule such as arabinose or hexitol; it is of interest to synthetic biology due to its resistance to nucleases and "orthogonality" to many systems, while still retaining the capacity for base pairing. The Cas9 protein, at least in its commonly used form, is used to target a particular sequence in a cell's DNA; to do this, it takes a short "guide" RNA strand complementary to the sequence to be targeted. We will be attempting to replace this guide RNA with an XNA molecule while still achieving successful genome targeting.
    The project will have two main goals. First, we will test for XNA compatibility in vitro. We will screen a number of different XNA variants (i.e. with different substitutions for the backbone sugars) for Cas9 compatibility, by determining whether the protein will bind an XNA "guide RNA" molecule in vitro; we will then test whether the resulting complex will successfully target the complementary DNA sequence. If XNA forms exhibit successful genome targeting, we will then move to in vivo testing, in which a Cas9 protein using an XNA guide (most likely introduced to the cell in the form of oligonucleotides) will be used as part of a genetic circuit.

More ideas:

We could also try guide RNAs that are only partially replaced by XNA (for example, XNA caps at the ends, or replacing only the DNA-binding section).

RNAi/miRNAs?

There do exist XNA polymerases, able to transcribe XNA from DNA (and, in one case, from XNA); there also exist XNA-dependent DNA polymerases enabling us to convert it back. However, many of the proteins involved in replication (helicase, topoisomerase, SSBPs, etc.) do not yet exist for XNAs, so sustained in vivo replication probably isn't something we want to try. Also, getting the XNA nucleotides into a cell for XNA synthesis would be difficult, since I doubt the cell would take them up naturally or that the existing kinases would be able to add phosphate groups (although there do exist general NTP transporters, which could potentially be modified to take up XNA triphosphates).

Some forms of XNA have been shown to work with RNAi.