CRISPR-inspired systems have been extensively developed for applications in genome editing and nucleic acid detection. Here, we introduce a CRISPR-based peptide display technology to facilitate ...customized, high-throughput in vitro protein interaction studies. We show that bespoke peptide libraries fused to catalytically inactive Cas9 (dCas9) and barcoded with unique single guide RNA (sgRNA) molecules self-assemble from a single mixed pool to programmable positions on a DNA microarray surface for rapid, multiplexed binding assays. We develop dCas9-displayed saturation mutagenesis libraries to characterize antibody-epitope binding for a commercial anti-FLAG monoclonal antibody and human serum antibodies. We also show that our platform can be used for viral epitope mapping and exhibits promise as a multiplexed diagnostics tool. Our CRISPR-based peptide display platform and the principles of complex library self-assembly using dCas9 could be adapted for rapid interrogation of varied customized protein libraries or biological materials assembly using DNA scaffolding.
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•dCas9-peptide fusions are barcoded with unique sgRNAs for peptide library studies•Peptides are directed to sgRNA-complementary sequences on a template DNA microarray•dCas9-peptide mixtures self-assemble on microarrays in a technique called PICASSO•PICASSO enables quantitative antibody binding characterization and epitope mapping
To facilitate custom peptide library studies, Barber et al. fuse peptide collections to dCas9 barcoded with unique sgRNA sequences. They demonstrate that, from mixed pools, dCas9-peptide fusions self-assemble to positions on a DNA microarray surface complementary to their sgRNA barcodes and they use the resulting microarrays to characterize antibody-epitope binding.
RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA ...would allow alteration and imaging of endogenous RNA transcripts analogous to CRISPR/Cas-based genomic tools, but most RNA targeting methods rely on incorporation of exogenous tags. Here, we demonstrate that nuclease-inactive S. pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells. We show that nuclear-localized RNA-targeting Cas9 (RCas9) is exported to the cytoplasm only in the presence of sgRNAs targeting mRNA and observe accumulation of ACTB, CCNA2, and TFRC mRNAs in RNA granules that correlate with fluorescence in situ hybridization. We also demonstrate time-resolved measurements of ACTB mRNA trafficking to stress granules. Our results establish RCas9 as a means to track RNA in living cells in a programmable manner without genetically encoded tags.
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•RNA-targeting Cas9 (RCas9) enabled recognition of endogenous, unmodified mRNAs•RCas9 did not influence mRNA abundance or amount of translated protein•Subcellular distribution of RCas9 was highly correlated with RNA-FISH•RCas9 revealed trafficking of mRNAs to stress granules in live cells
RNA-targeting Cas9 enables tracking of endogenous, untagged mRNA, establishing CRISPR/Cas9 as a programmable system to recognize RNA in live cells.
The cotton bollworm Helicoverpa armigera, is one of the world's major pest of agriculture, feeding on over 300 hosts in 68 plant families. Resistance cases to most insecticide classes have been ...reported for this insect. Management of this pest in agroecosystems relies on a better understanding of how it copes with phytochemical or synthetic toxins. We have used genome editing to knock out a cluster of nine P450 genes and show that this significantly reduces the survival rate of the insect when exposed to two classes of host plant chemicals and two classes of insecticides. Functional expression of all members of this gene cluster identified the P450 enzymes capable of metabolism of these xenobiotics. The CRISPR-Cas9-based reverse genetics approach in conjunction with in vitro metabolism can rapidly identify the contributions of insect P450s in xenobiotic detoxification and serve to identify candidate genes for insecticide resistance.
Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely ...employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.
The targeted deletion, replacement, integration or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically require double-strand DNA ...breaks (DSBs) that lead to undesired consequences, including uncontrolled indel mixtures and chromosomal abnormalities. Here we describe twin prime editing (twinPE), a DSB-independent method that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites. The two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the prime-editor-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled targeted integration of gene-sized DNA plasmids (>5,000 bp) and targeted sequence inversions of 40 kb in human cells. TwinPE expands the capabilities of precision gene editing and might synergize with other tools for the correction or complementation of large or complex human pathogenic alleles.
Cas9 and Cas12a are multidomain CRISPR‐associated nucleases that can be programmed with a guide RNA to bind and cleave complementary DNA targets. The guide RNA sequence can be varied, making these ...effector enzymes versatile tools for genome editing and gene regulation applications. While Cas9 is currently the best‐characterized and most widely used nuclease for such purposes, Cas12a (previously named Cpf1) has recently emerged as an alternative for Cas9. Cas9 and Cas12a have distinct evolutionary origins and exhibit different structural architectures, resulting in distinct molecular mechanisms. Here we compare the structural and mechanistic features that distinguish Cas9 and Cas12a, and describe how these features modulate their activity. We discuss implications for genome editing, and how they may influence the choice of Cas9 or Cas12a for specific applications. Finally, we review recent studies in which Cas12a has been utilized as a genome editing tool.
This article is categorized under:
RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications
Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs
RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
Swarts and Jinek describe how distinct structural features can determine the choice for Cas9 or Cas12a/Cpf1 for genome editing applications.
Abstract
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become a successful and promising technology for gene-editing. To ...facilitate its effective application, various computational tools have been developed. These tools can assist researchers in the guide RNA (gRNA) design process by predicting cleavage efficiency and specificity and excluding undesirable targets. However, while many tools are available, assessment of their application scenarios and performance benchmarks are limited. Moreover, new deep learning tools have been explored lately for gRNA efficiency prediction, but have not been systematically evaluated. Here, we discuss the approaches that pertain to the on-target activity problem, focusing mainly on the features and computational methods they utilize. Furthermore, we evaluate these tools on independent datasets and give some suggestions for their usage. We conclude with some challenges and perspectives about future directions for CRISPR–Cas9 guide design.
•CRISPR-Cas9 can inhibit HIV replication.•CRISPR-Cas9 can permanently inactivate latent HIV.•CRISPR-dCas9 systems can reactivate latent HIV.•Novel CRISPR-Cas based gene therapy strategies against HIV ...are feasible.
In bacteria and archaea, the clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas) confer adaptive immunity against exogenous DNA elements. This CRISPR-Cas system has been turned into an effective tool for editing of eukaryotic DNA genomes. Pathogenic viruses that have a double-stranded DNA (dsDNA) genome or that replicate through a dsDNA intermediate can also be targeted with this DNA editing tool. Here, we review how CRISPR-Cas was used in novel therapeutic approaches against the human immunodeficiency virus type-1 (HIV-1), focusing on approaches that aim to permanently inactivate all virus genomes or to prevent viral persistence in latent reservoirs.
Background. The fragrance terpene geraniol forms sensitizing compounds via autoxidation and skin metabolism. Geranial and neral, the two isomers of citral, are the major haptens formed in both of ...these activation pathways.
Objectives. To investigate whether testing with oxidized geraniol detects more cases of contact allergy than testing with pure geraniol.
Patients and methods. The pattern of reactions to pure and oxidized geraniol, and metabolites/autoxidation products, was studied to investigate the importance of autoxidation or cutaneous metabolism in contact allergy to geraniol. Pure and oxidized geraniol were tested at 2.0% petrolatum in 2227 and 2179 consecutive patients, respectively. In parallel, geranial, neral and citral were tested in 2152, 1626 and 1055 consecutive patients, respectively.
Results. Pure and oxidized geraniol gave positive patch test reactions in 0.13% and 0.55% of the patients, respectively. Eight of 11 patients with positive patch test reactions to oxidized geraniol also reacted to citral or its components. Relevance for the positive patch test reactions in relation to the patients' dermatitis was found in 11 of 14 cases.
Conclusions. Testing with oxidized geraniol could detect more cases of contact allergy to geraniol. The reaction pattern of the 14 cases presented indicates that both autoxidation and metabolism could be important in sensitization to geraniol.
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.
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•Cas9 can be circularly permuted (Cas9-CP) for optimized fusion protein construction•Cas9 circular permutation enables the engineering of protease-activated ProCas9s•ProCas9s can sense and respond to protease activity, such as during viral infection•Cas9-CPs provide a toolkit of safer and more efficient genome-modifying enzymes
Programmable Cas9 variants with improved functionality are generated by protein engineering—including versions that work as protease sensors responding to viral infection.
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