Based on your itinerary:
For Rome, the Jubilee will cause some extra crowds, but it's worth it because you'll see the Holy Door open in St. Peter's basilica, which happens only every 25 years. I'd make an entire day out of St. Peter, the Vatican Museum, and the Sistine Chapel. You won't even get through the entire museum in one day but pick the things you like and it will be a nice full day. I'd also check out Castel Sant'Angelo while you're on that side of the river because it's a beautiful castle right across the street from St. Peter's.
Colosseum and the Forum are right next to each other and easy to tour during the same outing. There are also the Traian markets right there which are often overlooked but very cool to see. While you're in the area go take a picture sticking your hand into the "Bocca della Verita'" (mouth of truth, which legend has it will close and crush your hand if you're a liar). Around the corner is a little church that has the tomb of emperor Adrian and the skull of St. Valentine, and right across are the temples of Portunus and Hercules the Victor. They're really small and often overlooked, but are actually the two best preserved buildings from that era. Go eat at "Osteria I Clementini" right by the Colosseum, it's owned by the mom of one of my high school friends and has legit Roman food despite it's location in a touristy area. It was my weekend hangout for years before hitting the bars.
Around the Pantheon make sure to have an espresso at "St. Eustacchio" (their quirk is it comes pre-sugared, so make sure to let them know if you want it black) and a gelato at Giolitti, which is still the best outfit in town for it, or at least top 3. Or head across to Piazza Navona, which is worth it on it's own for Bernini's fountain, and have a "tartufo nero" ice cream at "Tre Scalini". It's a ball of chocolate ice cream wrapped in shaved chocolate chunks to give it the appearance of a black truffle, with a candied cherry inside. If you can get reservations at Armando by the Pantheon it's a great traditional restaurant as well. The #2 best pizza in town is around there too: "Pizzeria Baffetto". It gets crowded so go early, but is totally worth it. My absolute favorite one is kind of far away from the typical tourist spots, but this one is second best and not too far behind. If you're Catholic I also recommend going up the holy stairs by the San Giovanni in Laterano basilica. Supposedly they were brought over from Jerusalem and Jesus himself climbed them. For Catacombs I'd head over to the Appian way and do the ones of San Callisto and San Sebastiano. They're right next to each other. There's also the "Critpa dei Cappuccini" which is also in the center and right next to a metro station if you're into Catacombs. It's a bit creepy in true Capuchin monk fashion, but the bones and skulls are laid out in an artistic and breathtaking way.
Since you're surprising the wife I would also take her to one of the romantic lookout spots over the city. You can either go to the Orange Garden right by the Circus Maximus, or the Terrazza del Pincio at Villa Borghese. Over there you'll find the Casina Valadier which is one of the hottest date spots if you're into fine dining, or just pop in for an aperitivo. 4-5 days in Rome you can do a lot, let me know if you need more detailed info about anything or other recommendations.
I would avoid driving in Rome. It's chaos and the traffic is horrific. It will put even the largest US city that claims they have the worst traffic to shame. Plus finding parking adds a whole new level of PITA to your itineraries. You can take the Leonardo Express from the airport to Termini central station, and ride the metro from there. Stay in a hotel in the center and grab a car when it's time to get out.
From there drive up to Cortona. To me it's worth a detour to Perugia along the way to visit a lovely medieval city that is so often overlooked. You will find very few tourists and it's a great place to walk around. Bonus if you go during truffle season because that's one of the regions they find them and you can get delicious truffle dishes for cheap almost anywhere. And I'm talking shockingly cheap compared to US prices. You can also tour the Perugina chocolate factory (where they make Baci) if you're into that, or visit the nearby town of Assisi which is also super picturesque, and see where St. Francis started it all with the order of Franciscan Friars.
It's a close drive to Florence from there. I personally think you can visit it in a day unless you're really into Renaissance art and want to spend some time at the Uffizzi galleries. You absolutely must try a Fiorentina steak, which is a 3lb T-bone cooked 5 min on each side and 15 min standing up on the bone on a wood fire. Only served rare and you will be asked to leave if you order anything other than rare. There's also the question of Antico Vinaio. People will line up for hours to get a sandwich there, and they are good, but I personally think Fratellini or Fettunta are equally good or even better for much less hassle. Speaking of lines, the affogato at Vivoli Gelateria exploded on social media recently. If it's not too much of a wait, I'd recommend that too.
I personally don't have much experience in Venice. I subscribe to the mantra of "visit it once to say you did and that's good enough". I do remember Murano was fantastic and recommend doing a glass blowing demonstration. If you're driving stay in a hotel on the mainland and ride a boat or water taxi into the island. They're charging a tourist fee now BTW. Doing all the activities in Venice from my one time there, it's easiest to arrange in one package. We had a guy that organized all the tours and took us around on his boat. There's a guy that a lot of pilots use when we have layovers there on the 767, I'll ask one of my buddies to send me his contact. Everyone has had great experiences. Take some time to have some "cicchetti", the local version of tapas with an aperitivo.
On the way from Florence to Venice I would stop in Bologna and/or Modena. Again underrated destinations by Americans with some of the best food in Italy and home of all the motorsports from Italy (Ferrari, Lamborghini, Pagani, Ducati, Maserati, literally all of them). They all have museums, you can tour a Parmigiano Reggiano, prosciutto, or balsamic vinegar production facility, and even have a look at the late Pavarotti's house. I've been spending a lot of time in the region recently and go there every couple of months so let me know if it's something you're interested in, I can give much more detailed recommendations
