CRISPR/CAS9
CRISPR/CAS9 is the exciting new technology on the block, representing a revolution in the way that we perform genetic modification. As the name suggests, it is a two part system:
CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) - Contains multiple ~20 bp sequences of DNA separated by a repeating sequence of DNA. These sequences are transcribed to form an RNA strand known as crRNA and assembled with a stem-loop structured RNA strand (tracrRNA) to form a guide RNA molecule (gRNA). In its natural state as a bacterial immune system, the CRISPR array is responsible for remembering sequences of foreign bacteriophage DNA that may endanger the cell.
CAS9 (CRISPR associated protein 9) - An enzyme that assembles with one of the gRNA (guide RNA) sequence transcribed from the CRISPR array. The original CAS9 protein binds to double stranded DNA that complements the gRNA sequence and cuts through both backbones, leaving a double-stranded break (DSB). The cell is then left to repair the DSB through one of two possible repair pathways;
NHEJ: Non-Homologous End Joining: If no homologous sequence is nearby, the repair enzyme will fill the gap with random base pairs as it attempts to fix the DSB. In most cases this will lead to degenerate proteins and the disabling of any nearby genes, which makes it an effective knockout strategy. It is possible that the introduced random base pairs will somehow lead to a novel protein, which means that targeting and disabling the start codon is an efficient design choice. Close that reading frame before it even opens!
HDR: Homology Directed Repair: If an existing template for the correct DNA sequence is nearby (e.g. a homologous chromosome, or a plasmid with the correct template), then the cell will use a repair enzyme to align the two sequences to repair the break according to the homologous sequence. It is possible to introduce a slightly modified sequence flanked by long homologous sequences to trick the cell into repairing the sequence according to your design. However this will not work for larger sequences, let alone whole genes.
It is important to remember that the early generations of CRISPR/CAS9 only have the cutting function! We’ll dive into some of the more advanced and novel systems that have some repair functionality, but for the most part - Cas9 cuts and then repair is the job of the host!
CRISPR/CAS9 is unique from previous genetic modification technologies mentioned in this guide in two distinct ways;
In-Vivo: CRISPR/CAS9 can be used to modify cells while they are still alive. Most protocols for genetic modification require lysis of the host to extract DNA for modification, which is subsequently inserted into a new host of the same species. These techniques will never work for modifying higher order lifeforms.
Retargetable: It can be recoded to match any target you desire. Restriction Enzyme digest is highly specific. Golden Gate Cloning and Gibson Assembly are more targetable, but only work on extracted plasmid targets. Integrative plasmids can insert DNA into the genome, but it’s inserted randomly and your modification can knock out important genes.
CRISPR/CAS9 can make retargetable modifications to plasmids or the genome, but it is very limited in its ability to insert larger gene sequences. In its current state it is ideal for single-base pair substitutions and knock-out experiments. However this is subject to change at any point, as labs across the planet race to create the newest, fanciest CAS9-derived proteins. The best is yet to come for this technology.
Note: As with all GM protocols described on this website, consult your national regulator to ensure that you aren’t breaking any laws. In Australia, CRISPR/CAS9 must be performed in a PC1+ space with the door closed and is limited to the exempt organisms list.
Target Selection
Step 1: Target Sequence
The first critical decision of a CRISPR/CAS9 experiment is to determine a target sequence that you wish to modify. You can use BLAST to search out specific genes in the genome of your target organism to find the sequence of a target gene. Download and attempt to annotate the sequence.
Step 2: Knockout or Edit?
Once you have a target, you need to decide whether you’re planning to just knockout the gene (NHEJ) or whether you wish to attempt a targeted edit (HDR). Note that the former is easy while the latter is extremely difficult.
Knockout: You can introduce the cut anywhere in the gene and it will likely knock it out, however you should be hunting down a target sequence near the 5’ end of the reading frame. This will prevent any unexpected consequences from malformed protein. Highlight ~30 bp around the area of interest.
Edit: If you’re attempting an edit, you should be hunting down a target sequence at the location that you wish to edit. Once you find the part of the gene that you wish to modify, highlight ~30 bp around the area of interest. Annotate the 1-2 base pairs that you wish to replace on your unmodified sequence and create a seperate ‘edited’ sequence document with the modification. This will be important for designing the homology template.
Step 3: PAM
The PAM sequence (NGG) is a three base-pair sequence critical to the targeting of Cas9. Search through the highlighted area downstream (in the 3’ direction) of your gene for the closest “NGG”. Top strand (sense) or bottom strand (anti-sense) is fine. Since N can be anything, you’re actually just looking for a “GG”. I’m unsure why they don’t just make the PAM sequence GG, maybe someone will explain it to me one day.
If it turns out that your PAM sequence is on the antisense strand, consider flipping the entire sequence (swaps sense/antisense) to make it easier to keep track of which strand is which.
The double stranded cut will occur 3-4 base pairs upstream (in the 5’ direction) of the PAM sequence.
Knockout: Anywhere is probably fine, just make sure that the cut is still occurring somewhere within the gene.
Edit: The PAM will need to be very close to the base pairs that you wish to modify. Ideally you can find a NGG sequence that is exactly 3-4 base pairs from those that you wish to replace.
If there is no NGG at all within the highlighted ~30 bp area, consider choosing a target elsewhere in the gene. Alternatively you can investigate alternative PAM CAS9s.
CRISPR/Cas9 System Selection
Which system fulfills the plans above…
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