Hey this is pretty good! Looks like you worked out how stupid stairs are in this game, most of the staircases seem to be optimised. I've noted down comments as I spotted them.
0) You lose frames throughout the run because names are not shortened, at least shortening Isaac's name is worth it
1) vs Bandits in Vault, you should take damage on Isaac so you can use Cure on him later in Goma cave. It would have saved you using two additional moves (~4s) after curing Garet to drop PP below 5 (you had 8 PP so 1 cure would drop that to 5PP)
2) Why did you leave Vault going through the south exit? All my tests indicated that going top was generally faster (unless I'm really bad at timing)
3) In Bilibin cave seems like you could let the "get item" animation last an additional frame to save attack cancelling in the next fight?
4) The second fight in Bilibin cave can't be optimal, way too many attack cancels. Use the get item RNG to manipulate a better path.
5) Hit three encounters on the way to Imil, can get that down to two with a better seed. There's also some sub-optimal movement you can use going around the corner to Imil to minimise the encounter counter which might be helpful
6) What happened when you moved the snowman in Imil? It didn't fall down in my movie, is that just an emu bug?
7) I'm surprised going the right staircase at Mercury entrance is faster, I thought left would clearly be better :P
8) Your retreat warp after beating the lizard fighter looked unoptimised, since you can do the warp lower and walk diagonally to minimise movement frames. I could be wrong about this though so I'm curious to know if you did this intentionally.
9) The Saturos fight was cool. I like using Isaac to heal after the Psynergy Stone. Shame that it doesn't save any time at Tret though.
10) I'm almost positive that skipping the first retreat warp in Tret will save time over this route. In RTA we can't control RNG so the retreat warp saves time on average, while in TAS it should be faster to manipulate fights to drop HP (and hence PP) and get optimal escape luck.
11) Another thing, you can actually do Tret backwards in RTA and I wonder if doing Tret backwards or using the early OOB would save time in a TAS setting (it loses something like 10-30 seconds in RTA the last time I tested it with RTA strats). In the second room of Tret take the left exit and you can get OOB there, you can skip right to Breeze in that OOB saving you climbing the tree. If you can find the right door this should be faster with TAS strats.
12) You use Cybele against the Killer Ape, usually we try to avoid this at all costs because it takes forever. Is there no way to avoid it in TAS?
13) You get a fight between Mogall Forest and Xian, I would suggest you add some RNG frames from one of the summon animations to avoid this encounter. It's certainly possible.
14) The door you used to skip Altin is not optimal in TAS. Its the only door you can hit reliably in RTA, but there is a door which takes you to the left exit which I could only ever reliably hit in a TAS setting. That saves a lot of time since you skip the Hama cutscene when you exit the right side. I can't find where I wrote down the coordinates for the door unfortunately :( but I do know that the door exists!
15) The manticore skip seemed good, but I wonder if you tested a S&Q-less skip to Manticore? You can enter reveal, go into retreat mode, then leave reveal to glitch out the Lamakan map. Slightly to the left of where you do the normal retreat warp there is a sinkhole embedded into the wall. If you fall into the sink hole, run from the enemy, you will get put at (0,0) and just need to press A to fight the manticore. It's definitely slower in RTA because Lamakan is hell, but it might be faster in a TAS setting.
16) I wonder if you could enter/exit Lamakan after getting vine to dodge an encounter?
17) In the ship menu you can at least 1 frame by doing the menu when you give the lucky charm to the captain instead of doing it before the fight.
18) Getting ground after altmiller is faster in a TAS setting. We get ground before Altmiller in RTA to make the RNG more consistent and hence faster on average. You don't have to worry about that so you should get ground after.
19) The post-ship part of the game is quite different to RTA so I don't have as many comments, aside from questioning the amount of saving and reseting you do. While I appreciate why you do that (more than the average person at least!) it would seem that more attack cancelling/grn manipulation could be used to get runs saving the reset.
20) Why do you skip the Gaia Blade? There doesn't seem to be a good explanation for this. Of course, you're not getting it for the attack power rather the +Venus power which gives judgement a nice buff which my logic tells me would save at least one action on Menardi/Saturos and FD which could compensate for the time taking to get it.
21) The S&M/FD fights are really long. I know in Any% that these fights are longer than 100% (which gets both down in 9 turns in RTA) but there is probably some rerouting you can do to speed this up.
All in all this was really fun to watch :) great job!