CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification

Abstract

The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.

Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

“…The completed reactions were then run on 6% TBE polyacrylamide gels for 1 hr at 50V and visualized with SYBRGold stain. vector pCF820, encoding a SpyCas9 U6-sgRNA cassette and an EF1a driven, human codon optimized mCherry2 marker (40), was based on the pCF221 vector (35). To make the backbone more efficient and increase viral titers, the f1 bacteriophage origin of replication and bleomycin resistance marker were removed, as has previously been done for pCF525 (19).…”

“…4A). Using HEK-RT1 genome editing reporter cells (35,36) -a monoclonal HEK293T-based cell line expressing a doxycycline-inducible GFP reporter -lentiviral constructs encoding the AcrIIA proteins or mTagBFP2, and SauCas9 or SpyCas9, were sequentially transduced and stable integrants selected. The resulting cell lines were then further transduced with a vector expressing a GFP-targeting or negative control sgRNA and an associated mCherry fluorescence marker.…”

Cancer-specific loss of TERT activation sensitizes glioblastoma to DNA damage

“…To generate monoclonal knockout cell lines, sgRNAs and Cas9 were expressed from either the pCF120 or pCF123 vectors. In brief, the plasmid vector pCF120, expressing a U6 promoter driven sgRNA and an EF1-alpha short (EFS) promoter driven Cas9-P2A-Hygro cassette, was derived from pX459 60 by replacing the chicken beta-actin promoter with an EFS promoter from pCF204 61 , swapping the T2A-Puro cassette with a P2A-Hygro cassette, and exchanging the guide RNA scaffold for a more efficient variant 62 . The plasmid vector pCF123, expressing a U6 driven sgRNA and an EFS driven Cas9-T2A-Puro cassette, was generated based on pX459 60 by replacing the chicken beta-actin promoter with an EFS promoter and exchanging the guide RNA scaffold for a more efficient variant 62 .…”

“…For CRISPR-Cas9-mediated competitive proliferation assays, single-guide RNAs (sgRNAs) were expressed from the previously established pCF221 lentiviral vector and Cas9 from the pCF226 lentiviral vector 61 . For low-level stable overexpression of GABPB2 (NCBI gene ID: 126626) and mTagBFP2 (negative control), we first modified the pCF525-mTagBFP2 lentiviral vector 63 , encoding an EF1a-Hygro-P2A-mTagBFP2 cassette, by replacing the strong EF1-alpha promoter with a weak EF1-alpha short (EFS) promoter.…”

“…Lentiviral particles were produced in HEK293T cells using polyethylenimine (PEI; Polysciences #23966) based transfection of plasmids, as previously described 61 . In brief, lentiviral vectors were co-transfected with the lentiviral packaging plasmid psPAX2 (Addgene #12260) and the VSV-G envelope plasmid pMD2.G (Addgene, #12259).…”