Guide RNA functional modules direct Cas9 activity and orthogonality.
The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner and has been widely used for programmable genome editing. Cas9 activity is dependent on interactions with guide RNAs, and evolutionarily divergent Cas9 nucleases have been shown to work orthogonally. However, the molecular basis of selective Cas9:guide-RNA interactions is poorly understood. Here, we identify and characterize six conserved modules within native crRNA:tracrRNA duplexes and single guide RNAs (sgRNAs) that direct Cas9 endonuclease activity. We show the bulge and nexus are necessary for DNA cleavage and demonstrate that the nexus and hairpins are instrumental in defining orthogonality between systems. In contrast, the crRNA:tracrRNA complementary region can be modified or partially removed. Collectively, our results establish guide RNA features that drive DNA targeting by Cas9 and open new design and engineering avenues for CRISPR technologies.
Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA.
Alexandra E
AE
Briner
Caribou Biosciences, Inc., Berkeley, CA 94710, USA.
Paul D
PD
Donohoue
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA; Chemical Engineering Department, Faculty of Engineering, Cairo University, Giza 12613, Egypt.
Ahmed A
AA
Gomaa
Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA.
Kurt
K
Selle
Caribou Biosciences, Inc., Berkeley, CA 94710, USA.
Euan M
EM
Slorach
Caribou Biosciences, Inc., Berkeley, CA 94710, USA.
Christopher H
CH
Nye
Caribou Biosciences, Inc., Berkeley, CA 94710, USA.
Rachel E
RE
Haurwitz
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA. Electronic address: cbeisel@ncsu.edu.
Chase L
CL
Beisel
Caribou Biosciences, Inc., Berkeley, CA 94710, USA. Electronic address: andy.may@cariboubio.com.
Andrew P
AP
May
Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA. Electronic address: rbarran@ncsu.edu.
Rodolphe
R
Barrangou
D001426:Bacterial Proteins / Q000737:chemistry*; D000076987:CRISPR-Associated Protein 9; D064130:CRISPR-Associated Proteins / Q000737:chemistry*; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000737:chemistry*; D053837:DNA Cleavage; D004720:Endonucleases / Q000737:chemistry*; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure
D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases