Choose the exon(s) you want to target
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We will use the gene appa as an example.
Look at the transcripts in the Genome Browser track, e.g. appa-203, appa-205, etc.
Pay attention to the direction of the gene. e.g. appa is reverse, because the transcripts are written as < appa-203. If it was forward, they would be written as > appa-203.
If the gene is forward, left-most exon is exon 1, i.e. read the transcript from left to right.
If the gene is reverse, right-most exon is exon 1, i.e. read the transcript from right to left.
Choose a few exons that you may want to target. If making F0 knockouts using the method in 10.7554/eLife.59683 you will (ideally) target three separate exons. Try to find 3–5 exons that make good targets. Try to target (roughly in order of priority):
Exons that are common to all/most transcripts. Ignore transcripts that are not protein-coding (e.g. retained intron or processed transcript). Transcripts do not always overlap so it is not always possible to target exons common to all transcripts, but do the best you can. If doing F0 knockouts, try to have each transcript targeted by at least 1 gRNA.
Early exons. Make sure to read to the transcript in the right direction for this (see above).
Asymmetrical exons (see below).
Not exon 1, as there may be alternative start codons towards the beginning of the transcript.
It is rare to find more than 2 exons that fulfill all criteria, but follow as guidance to prioritise exons.
About 3. asymmetrical exons:
An exon is symmetrical if its length is a multiple of 3, i.e. it has a round number of codons.
An exon is asymmetrical if its length is not a multiple of 3, i.e. it does not have a round number of codons.
Exon skipping is a common compensatory mechanism where the cell splices out an exon which contains a mutation. This could cancel your frameshift mutation by skipping the mutated exon altogether.
Skipping a symmetrical exon keeps the reading frame intact. For example, skipping a 300-bp exon deletes exactly 100 codons, it does not create a frameshift.
However, skipping an asymmetrical exon shifts the frame of the mRNA after the skipped exon. For example, skipping a 299-bp exon deletes 99 codons and 2 bp of the next codon, so shifts the reading frame.
Therefore, targeting asymmetrical exons is a strategy that nullifies exon skipping as a possible compensatory mechanism. For example, say you generate a 2-bp deletion in an asymmetrical (299-bp long) exon. If the exon is not skipped, the 2-bp deletion shifts the reading the frame; if the exon is skipped, the 299-bp deletion shifts the reading frame. Either way, you created a frameshift mutation.
This criterion is not essential when generating F0 knockouts with 3 gRNAs. As another two exons should also carry mutations, it is unlikely that skipping one mutated exons is sufficient to make an mRNA that encodes a functional protein. However, it is more important to target an asymmetrical exon when generating a stable knockout line with a unique gRNA.
Choose the longest protein-coding transcript as reference for numbering the exons. For this, see length of the protein in the transcript table, e.g. transcript appa-202 codes for the longest protein, it is 682-amino acid long.
In the Genome Browser, you can also click on an exon to get its number.
Open the page of the transcript you chose as reference; e.g.
Go to Sequence > Exons, on the left column
Scroll down to the table. ENSDARE… are exon IDs. Look at the Length column.
Is the length of the exon a multiple of 3? Then the exon is symmetrical.
Is the length of the exon not a multiple of 3? Then the exon is termed asymmetrical.
Note; the first and last exon may include a UTR. Symmetrical/asymmetrical is only meaningful for protein-coding exons, so do not count the length of the UTR.
The first exon includes a 5'-UTR (in orange). Its length (217 bp) includes this UTR, so you need to substract the length of the UTR from 217 bp. Here the UTR is 60 bp, so the protein-coding part of this exon is 217 − 60 = 157 bp, i.e. the exon is asymmetrical.
You do not want to count the length of the UTR, as the ribosome does not read it.
Exon 5 has a UTR (orange), but it is counted in the length. So copy-paste just the protein-coding (blue) sequence and count the number of characters.
Following the criteria above, select at least one (for stable knockout) or at least three (for F0 knockout) exons that make good targets.
e.g. following appa-202 as reference transcript, I would select exon 6/19, exon 8/19, exon 15/19. Note that there is no exact answer.