Immune　checkpoint　blockade　based　on　antibodies　has　shown　great　clinical　success　in　patients,　but　the　transitory　working　manner　leads　to　restricted　therapeutic　benefits.　Herein,　a　genetically　engineered　adenovirus　is　developed　as　the　vector　to　deliver　CRISPR/Cas9　(sgCas9-AdV)　to　achieve　permanent　PD-L1　gene　editing　with　efficiency　up　to　78.7%　exemplified　in　Hepa　1-6　liver　cancer　cells.　Furthermore,　the　sgCas9-AdV　is　loaded　into　hydrogel　made　by　silk　fiber　(SgCas9-AdV/Gel)　for　in　vivo　application.　The　silk-gel　not　only　promotes　local　retention　of　sgCas9-AdV　in　tumor　tissue,　but　also　masks　them　from　host　immune　system,　thus　ensuring　effectively　gene　transduction　over　9　days.　Bearing　these　advantages,　the　sgCas9-AdV/Gel　inhibits　Hepa　1-6　tumor　growth　with　100%　response　rate　by　single-dose　injection,　through　efficient　PD-L1　disruption　to　elicit　a　T　cell-mediated　antitumor　response.　In　addition,　the　sgCas9-AdV/Gel　is　also　successfully　extended　into　other　refractory　tumors.　In　CT26　colon　tumor　characterized　by　poor　response　to　anti-PD-L1,　sgCas9-AdV/Gel　is　demonstrated　to　competent　and　superior　anti-PD-L1　antibody　to　suppress　tumor　progression.　In　highly　aggressive　orthotopic　4T1　mouse　breast　tumor,　such　a　therapeutic　paradigm　significantly　inhibits　primary　tumor　growth　and　induces　a　durable　immune　response　against　tumor　relapse/metastasis.　Thus,　this　study　provides　an　attractive　and　universal　strategy　for　immunotherapy.　©　2023　The　Authors.　Advanced　Science　published　by　Wiley-VCH　GmbH.