The Search Engine: Guide RNA Function

If the targeting process involves base pairing (A to U, C to G), then the strands are held together by weak electrostatic attractions. If these attractions are non-covalent, then they are hydrogen bonds.

If the enzyme relies entirely on the gRNA to find its destination, then without a guide, it is "blind." If it cannot find a target, then no specific or controlled DNA editing can occur.

If the guide finds the spot, the DNA must open so the guide can pair with it. If the PAM is confirmed, then the enzyme triggers its cutting action, resulting in a break in both strands of the DNA.

If the crRNA holds the "code" but cannot bind to Cas9 alone, then it needs a helper RNA. If this helper RNA facilitates the binding of the whole complex, then it is the tracrRNA.

If there are billions of bases in a genome, then finding a truly unique 20-base sequence is difficult for a human. If software can scan the genome for potential off-targets, then it ensures the gRNA is as specific as possible.

If the gRNA is programmed with the "address" for the insulin gene, then the enzyme will only be directed to that specific location. If the targeting is successful, then the cut is localized to that gene.

If the targeting sequence changes for every gene, then another part must remain the same so the protein can hold onto it. If this "handle" is universal for all gRNAs, then it is the scaffold.

If scientists use a "dead" Cas9 that doesn't cut, then the gRNA can still lead it to a gene. If the gRNA carries a fluorescent tag or a gene activator, then it can be used for imaging or turning genes on.

If the sgRNA is a functional tool, then it needs a "search" end (targeting) and a "handle" end (scaffold). Since it is RNA, it contains Uracil rather than Thymine and is a single-strand molecule.

If a GPS uses coordinates to find a house on a map, then the gRNA uses base sequences to find a gene in the DNA. If both systems provide precise navigation to a destination, then the analogy is accurate.

If the Cas9 protein is unable to identify a target sequence on its own, then it requires a navigation tool. If the gRNA contains a sequence that matches the target, then it successfully directs the enzyme to the correct spot.

If the Cas9 protein is a universal "engine" and the gRNA is the "direction," then only the gRNA needs to change. If you swap the 20-base sequence in the gRNA, then you have reprogrammed the entire system.

If a gRNA matches a non-target sequence by 18 out of 20 bases, then it may accidentally bind there. If there is too much Cas9, the likelihood of these "near-miss" bindings increases, causing unintended cuts.

If the Cas9 protein needs a "handhold" to distinguish the target from the cell's own DNA, then it looks for a specific 2-3 base motif. If this PAM sequence is present, then the guide can successfully lock the enzyme onto the DNA.

If the gRNA mimics the "memory" stored in the bacterial genome, then it uses the unique segments between repeats. If these unique segments are called spacers, then that is the name of the targeting sequence.

If the RNA only provides the location and not the ability to cut, then it is useless alone. If the gRNA must form a complex with the Cas9 protein to be functional, then it is not independent.

If the natural system requires a "map" and "scissors," then the crRNA/tracrRNA act as the map and Cas9 as the scissors. DNA polymerase and ribosomes are involved in other cell processes, not the CRISPR guide system.

If a sequence is too short, then it might appear in many random places in the genome. If a sequence of approximately 20 bases is unique enough to appear only once, then the gRNA can target a single gene accurately.

If the system is simplified for easier use in gene editing, then the two natural RNA components are fused into one. If they are fused into a single strand, then it is known as a single-guide RNA or sgRNA.

If the gRNA sequence is 5-GATTACA-3, then it will seek out a DNA sequence that matches its complementary bases. If these bases bond together, then the gRNA has successfully located the target.