[en] CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery of genetically encoded CRISPR components remains challenging and sustained Cas9 expression correlates with higher off-target activities, which can be reduced via Cas9-protein delivery. Here we demonstrate that baculovirus, alongside its DNA cargo, can be used to package and deliver proteins to human cells. Using protein-loaded baculovirus (pBV), we demonstrate delivery of Cas9 or base editors proteins, leading to efficient genome and base editing in human cells. By implementing a reversible, chemically inducible heterodimerization system, we show that protein cargoes can selectively and more efficiently be loaded into pBVs (spBVs). Using spBVs we achieved high levels of multiplexed genome editing in a panel of human cell lines. Importantly, spBVs maintain high editing efficiencies in absence of detectable off-targets events. Finally, by exploiting Cas9 protein and template DNA co-delivery, we demonstrate up to 5% site-specific targeted integration of a 1.8 kb heterologous DNA payload using a single spBV in a panel of human cell lines. In summary, we demonstrate that spBVs represent a versatile, efficient and potentially safer alternative for CRISPR applications requiring co-delivery of DNA and protein cargoes.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Capin, Julien ✱; School of Biochemistry, University of Bristol, 1 Tankard's Close, Bristol BS8 1TD, UK
Harrison, Alexandra ✱; School of Biochemistry, University of Bristol, 1 Tankard's Close, Bristol BS8 1TD, UK
Raele, Renata A; School of Biochemistry, University of Bristol, 1 Tankard's Close, Bristol BS8 1TD, UK
Yadav, Sathish K N; School of Biochemistry, University of Bristol, 1 Tankard's Close, Bristol BS8 1TD, UK
Baiwir, Dominique ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques
Mazzucchelli, Gabriel ; Université de Liège - ULiège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)
Quinton, Loïc ; Université de Liège - ULiège > Département de chimie (sciences) > Chimie biologique
Satchwell, Timothy J; School of Biochemistry, University of Bristol, 1 Tankard's Close, Bristol BS8 1TD, UK
Toye, Ashley M; School of Biochemistry, University of Bristol, 1 Tankard's Close, Bristol BS8 1TD, UK
Schaffitzel, Christiane ; School of Biochemistry, University of Bristol, 1 Tankard's Close, Bristol BS8 1TD, UK
Berger, Imre ✱; School of Biochemistry, University of Bristol, 1 Tankard's Close, Bristol BS8 1TD, UK ; School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, UK ; Max Planck Bristol Centre for Minimal Biology, Cantock's Close, Bristol BS8 1TS, UK
Aulicino, Francesco ✱; School of Biochemistry, University of Bristol, 1 Tankard's Close, Bristol BS8 1TD, UK
✱ These authors have contributed equally to this work.
Language :
English
Title :
An engineered baculoviral protein and DNA co-delivery system for CRISPR-based mammalian genome editing.
ERC - European Research Council MRC - Medical Research Council University of Bristol Max Planck-Bristol Centre for Minimal Biology
Funding text :
European Research Council (ERC) Advanced Grant [DNADOCK, Project No. 834631]; GE Healthcare [Discovery Research Grant to I.B.]; Medical Research Council Grant [MR/V010506/1 to A.M.T. and T.J.S.]; BrisSynBio, a
BBSRC/EPSRC Research Centre for Synthetic Biology at the
University of Bristol [BB/L01386X/1 to I.B.]; EPSRC and
BBSRC Centre for Doctoral Training in Synthetic Biology
[EP/L016494/1 to J.C.]; Max Planck Centre for Minimal Biology. Funding for open access charge: University of Bristol. European Research Council (ERC) Advanced Grant [DNA- DOCK, Project No. 834631]; GE Healthcare [Discov- ery Research Grant to I.B.]; Medical Research Council Grant [MR / V010506 / 1 to A.M.T . and T .J.S.]; BrisSynBio, a BBSR C / EPSR C Research Centre for Synthetic Biology at the University of Bristol [BB / L01386X / 1 to I.B.]; EPSRC and BBSRC Centre for Doctoral Training in Synthetic Biology [EP / L016494 / 1 to J.C.]; Max Planck Centre for Minimal Bi- ology. Funding for open access charge: University of Bristol.
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