Re-engineered RNA-Guided FokI-Nucleases for Improved Genome Editing in Human Cells.
Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 enables us to generate targeted sequence changes in the genomes of cells and organisms. However, off-target effects have been a persistent problem hampering the development of therapeutics based on CRISPR/Cas9 and potentially confounding research results. Efforts to improve Cas9 specificity, like the development of RNA-guided FokI-nucleases (RFNs), usually come at the cost of editing efficiency and/or genome targetability. To overcome these limitations, we engineered improved chimeras of RFNs that enable higher cleavage efficiency and provide broader genome targetability, while retaining high fidelity for genome editing in human cells. Furthermore, we developed a new RFN ortholog derived from Staphylococcus aureus Cas9 and characterize its utility for efficient genome engineering. Finally, we demonstrate the feasibility of RFN orthologs to functionally hetero-dimerize to modify endogenous genes, unveiling a new dimension of RFN target design opportunities.
Stem Cell and Regenerative Biology, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore 138672, Singapore.
Steven
S
Havlicek
Bioinformatics Core, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore 138672, Singapore.
Yang
Y
Shen
Endodermal Development and Differentiation Laboratory, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), Singapore 138672, Singapore.
Yunus
Y
Alpagu
Stem Cell and Regenerative Biology, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore 138672, Singapore.
Michaela B
MB
Bruntraeger
Stem Cell and Regenerative Biology, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore 138672, Singapore.
Nurdiana B M
NB
Zufir
Stem Cell and Regenerative Biology, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore 138672, Singapore.
Zhi Yi
ZY
Phuah
Bioinformatics Core, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore 138672, Singapore.
Zhiyan
Z
Fu
Endodermal Development and Differentiation Laboratory, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), Singapore 138672, Singapore.
Norris R
NR
Dunn
Stem Cell and Regenerative Biology, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore 138672, Singapore; Department of Biological Sciences, National University of Singapore, Singapore 119077, Singapore; School of Biological Sciences, Nanyang Technological University, Singapore 639798, Singapore. Electronic address: stantonl@gis.a-star.edu.sg.
Lawrence W
LW
Stanton
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism; D000072669:Gene Editing; D014644:Genetic Variation; D006801:Humans; D008954:Models, Biological; D009154:Mutation; D039904:Pluripotent Stem Cells / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D055503:Protein Multimerization; D000094704:RNA, Guide, CRISPR-Cas Systems; D000071701:Regulatory Factor X Transcription Factors / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D000071701:Regulatory Factor X Transcription Factors; C547964:Rfx6 protein, human; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific
CRISPR/Cas9; FokI; RNA-guided FokI nuclease; gene editing