One technique that I learned more about in BIOL 463 is CRISPR/Cas9, which stands for clustered regularly interspaced short palindromic repeats and CRISPR associated protein 9. Based on a mechanism of adaptive immunity in prokaryotes against viruses, CRISPR/Cas9 is used by researchers to edit genomes in cells. Essentially, the CRISPR/Cas9 system aims to induce site-specific double-stranded breaks in the genome of cells to allow homology directed repair after adding a DNA template consisting of the modified sequence which we wish to incorporate.
If someone were to learn how CRISPR/Cas9 works, I think the most difficult aspect of this technique to understand is that the single guide RNA (sgRNA) used to target the DNA sequence of interest must consist of both CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). Although the crRNA component of the sgRNA is what actually binds to target DNA by complimentary base pairing, the crRNA component is not sufficient to allow cleavage to occur by Cas9. As CRISPR/Cas9 is famous for its ability to allow site-specific modifications, students who first learn about CRISPR/Cas9 may become too focussed on the aspects that allow specific targeting, such as crRNA, and may overlook another aspect that is not involved in targeting DNA, but is necessary for the CRISPR/Cas9 system to work, such as tracrRNA, especially when the two components are part of a single RNA construct.
If I were to test a person’s understanding of how CRISPR/Cas9 works, I would ask: Why is a DNA template necessary to allow genome editing to occur through CRISPR/Cas9? In an answer, I would expect the person to acknowledge that without a DNA template, double-stranded breaks in the DNA will lead to non-homologous end joining, which results in the incorporation of random bases, and thus random mutations. Purposeful genome editing involves specific modification of sequences, rather than random mutagenesis. Therefore, a DNA template is necessary to allow specific modification of target DNA sequences.