Reproduction steps:
1. Open attached project "SortingLayerUpdate.zip" and create a new scene
2. In Project window, select "Prefab"
3. In Inspector window, go to Sprite Renderer -> Sorting Layer -> Add Sorting Layer...
4. Create a new Sorting Layer
5. Slot the newly created Sorting Layer between the "3" and "4" layers
6. In the Sprite Renderer of the Prefab, set the Sorting Layer to the new Sorting Layer
7. Save the project to update the Prefab
8. Open "Third Scene"
9. You should see a check mark in a red box.

Expected: Check mark in a red box
Actual: Red box

Note:
Without the fix, selecting the 4th Layer GameObject, and disabling and enabling the Sorting Group will give the expected result

Using the user's project and reproduction steps should not result in any scene differences for Gameplay scene.