Knost turned in a polished effort in ideal conditions at the Harbour Town Golf Links, carding two birdies on his outward nine and three more coming home to help him to a two-day total of nine-under 133 and a two-shot lead over Swede Carl Pettersson.
"One of my goals this year is to win," Knost, who had shared the overnight lead with Chad Campbell and Vaughn Taylor, said.
"I'm happy with how my game is and I'm happy with how I'm progressing. I like my chances and I love the way I'm playing right now."
Pettersson put himself into contention with a brilliant six-under 65 to sit alone in second, one stroke clear of Americans Harris English, with a 68, and two-time champion Boo Weekly, who moved into position for a Heritage hat-trick with a 66.
After an opening round of level par, Brandt Snedeker's title defence picked up steam with a scorching front nine that included six birdies.
The American stumbled on the inward nine with a pair of bogeys but still finished with a solid four-under 67 to join a group of four other players tied for seventh place on 138, five shots off the pace.
British world number one Luke Donald, who lost to Snedeker in a playoff last year, was also in battling mood, rebounding from an opening round 75 with a two-under 69 to just slip in just under the three-over cutline.
Donald needs a top eight finish this week to maintain his top ranking ahead of Northern Irishman Rory McIlroy.
American Jason Dufner, who had a share of the midway lead at the Masters last week, joined Donald on two-over 144 after signing for a bogey-free 66.
Padraig Harrington had been hoping to build on a solid eighth place showing at Augusta but the Irishman will have the weekend off after a one-over 72 left him one short of the cut.
Harrington was joined by Spanish Ryder Cup captain Jose Maria Olazabal, who returned a 73 to miss the cut by five shots.