ABSTRACT
CRISPR-mediated genome editing of primary human lymphocytes is typically carried out via electroporation, which can be cytotoxic, cumbersome and costly. Here we show that the yields of edited primary human lymphocytes can be increased substantially by delivering a CRISPR ribonucleoprotein mixed with an amphiphilic peptide identified through screening. We evaluated the performance of this simple delivery method by knocking out genes in T cells, B cells and natural killer cells via the delivery of Cas9 or Cas12a ribonucleoproteins or an adenine base editor. We also show that peptide-mediated ribonucleoprotein delivery paired with an adeno-associated-virus-mediated homology-directed repair template can introduce a chimaeric antigen receptor gene at the T-cell receptor α constant locus, and that the engineered cells display antitumour potency in mice. The method is minimally perturbative, does not require dedicated hardware, and is compatible with multiplexed editing via sequential delivery, which minimizes the risk of genotoxicity. The peptide-mediated intracellular delivery of ribonucleoproteins may facilitate the manufacturing of engineered T cells.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Humans , Mice , Animals , Gene Editing/methods , T-Lymphocytes/metabolism , Peptides/genetics , RibonucleoproteinsABSTRACT
The p19 viral suppressor of RNA silencing protein has useful applications in biotechnology due to its high affinity for binding to small RNAs such as small interfering RNAs (siRNAs). Also, its applications for the study and modulation of microRNAs are actively expanding. Here we demonstrate the successful site-specific incorporation of a photoactivatable unnatural amino acid, p-azido-l-phenylalanine (AzF), for cross-linking to RNA substrates into the p19 sequence. Incorporation of AzF was performed at three positions in the protein near the RNA binding site: K67, R115, and T111. Incorporation of AzF at position T111 of p19 did not affect the binding affinity of p19 for siRNAs and also showed nanomolar affinity for human microRNA miR-122. The affinity was less favorable with AzF incorporation at two other positions, suggesting the sensitivity of placement of the unnatural amino acid. Exposure of the T111AzF in complex with either siRNA or miRNA to ultraviolet light resulted in cross-linking of the protein with the RNA, but no cross-linking could be detected with the wild-type protein. Our results demonstrate that p19-T111AzF can be used for detection of small RNAs, including human miR-122, with high sensitivity and to irreversibly sequester these RNAs through covalent photo-cross-linking.
Subject(s)
MicroRNAs/analysis , RNA-Binding Proteins/metabolism , Viral Proteins/chemistry , Humans , Protein Binding , RNA, Small Interfering/analysis , RNA-Binding Proteins/chemistry , Sensitivity and SpecificityABSTRACT
Viral suppressors of RNA silencing (VSRSs) are a diverse group of viral proteins that have evolved to disrupt eukaryotic RNA silencing pathways, thereby contributing to viral pathogenicity. The p19 protein is a VSRS that selectively binds to short interfering RNAs (siRNAs) over microRNAs (miRNAs). Mutational analysis has identified single amino acid substitutions that reverse this selectivity through new high-affinity interactions with human miR-122. Herein, we report crystal structures of complexed p19-T111S (2.6 Å), p19-T111H (2.3 Å) and wild-type p19 protein (2.2 Å) from the Carnation Italian ringspot virus with small interfering RNA (siRNA) ligands. Structural comparisons reveal that these mutations do not lead to major changes in p19 architecture, but instead promote subtle rearrangement of residues and solvent molecules along the p19 midline. These observations suggest p19 uses many small interactions to distinguish siRNAs from miRNAs and perturbing these interactions can create p19 variants with novel RNA-recognition properties. DATABASE: Model data are deposited in the PDB database under the accession numbers 6BJG, 6BJH and 6BJV.
Subject(s)
Mutant Proteins/chemistry , RNA Interference , RNA, Small Interfering/chemistry , Tombusvirus , Viral Core Proteins/chemistry , Binding Sites/genetics , Cells, Cultured , Crystallization , Crystallography, X-Ray , Escherichia coli/cytology , Humans , Hydrogen Bonding , MicroRNAs/chemistry , Point Mutation , Protein Binding , Protein Engineering/methods , Protein Structure, Secondary , RNA, Double-Stranded , Viral Core Proteins/geneticsABSTRACT
Clustered regularly interspaced short palindromic repeats (CRISPR)-driven genome editing has rapidly transformed preclinical biomedical research by eliminating the underlying genetic basis of many diseases in model systems and facilitating the study of disease etiology. Translation to the clinic is under way, with announced or impending clinical trials utilizing ex vivo strategies for anticancer immunotherapy or correction of hemoglobinopathies. These exciting applications represent just a fraction of what is theoretically possible for this emerging technology, but many technical hurdles must be overcome before CRISPR-based genome editing technology can reach its full potential. One exciting recent development is the use of CRISPR systems for diagnostic detection of genetic sequences associated with pathogens or cancer. We review the biologic origins and functional mechanism of CRISPR systems and highlight several current and future clinical applications of genome editing.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Neoplasms/diagnosis , Neoplasms/genetics , Animals , HumansABSTRACT
Endogenously expressed noncoding RNAs are regulators of mRNA translation and affect diverse biological pathways spanning embryogenesis to cholesterol and fatty acid metabolism. Recently, microRNAs have become an important therapeutic target with strategies that employ oligonucleotides as both mimics and inhibitors of target microRNAs, successfully altering gene expression and cellular pathways in relevant contexts. However, delivery of these exogenous effectors remains a major challenge. Here, we present a method for evaluating noncoding RNA delivery using the viral suppressor of RNA silencing (VSRS) protein p19, optimized for cellular delivery of small RNAs. Using genetic code expansion technology, p-azidophenylalanine (AzF) was incorporated into a recombinant p19 protein and used to develop a fluorescence resonance energy transfer (FRET) sensor. AzF was used to attach FRET acceptor moieties using bioorthogonal chemistry. We show that this strategy not only gives rise to FRET signals that report on small RNA binding, but also allows for fluorescence quenching as well, convenient for measuring RNA release. We demonstrate the successful use of a modified version of the probe to track the delivery and release of small RNAs into mammalian cells. The results provide a basis for a further development of vehicles for small RNA delivery and release for intervening in noncoding RNA biology.
Subject(s)
Genetic Code , RNA, Small Interfering/administration & dosage , RNA-Binding Proteins/metabolism , Animals , Fluorescence Resonance Energy Transfer , MicroRNAs/metabolism , Protein Biosynthesis , RNA InterferenceABSTRACT
Despite the unparalleled therapeutic promise of genome editing, its curative power is currently limited by the substantial difficulty in delivering DNA-cutting enzymes to the cells in need of correction. A recent study demonstrates the potential for the delivery of pre-assembled genome-editing enzymes in the form of ribonucleoprotein complexes, which were used to rescue a mouse model of fragile X syndrome (FXS).
