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Boris Johnson's head of policy has resigned over the PM's false claim that Sir Keir Starmer failed to prosecute serial sex offender Jimmy Savile when he was director of public prosecutions.
Mr Johnson made the widely debunked accusation on Monday, sparking anger from MPs - including in his own party.
The PM backed down on Thursday, saying the Labour leader "had nothing to do personally with those decisions".
But Munira Mirza said he should have apologised for the misleading remarks.
In her resignation letter, published by The Spectator, she wrote: "You are a better man than many of your detractors will ever understand, which is why it is so desperately sad that you let yourself down by making a scurrilous accusation against the leader of the opposition."
A No 10 source has confirmed Ms Mirza's resignation to the BBC.
Mr Johnson faced criticism on Monday after a Commons debate where he accused Sir Keir of spending "most of his time" as director of public prosecutions (DPP) "prosecuting journalists and failing to prosecute Jimmy Saville", despite having no evidence to back up the claim.
At Wednesday's Prime Minister's Questions, the Labour leader then accused the PM of fuelling conspiracy theories with his remarks. Mr Johnson replied that Sir Keir had taken "full responsibility for what had happened on his watch".
By Thursday, Mr Johnson had appeared to back down, telling reporters he had not been talking about Sir Keir's "personal record" when he was DPP, adding: "I totally understand that he had nothing to do personally with those decisions.
"I was making a point about his responsibility for the organisation as a whole."
Asked about Mr Johnson's latest comments, Sir Keir told BBC Three Counties Radio: "If the PM thought that, he should have kept his mouth shut."