CRISPR technologies for genome, epigenome and transcriptome editing.

Our ability to edit genomes lags behind our capacity to sequence them, but the growing understanding of CRISPR biology and its application to genome, epigenome and transcriptome engineering is narrowing this gap. In this Review, we discuss recent developments of various CRISPR-based systems that can transiently or permanently modify the genome and the transcriptome. The discovery of further CRISPR enzymes and systems through functional metagenomics has meaningfully broadened the applicability of CRISPR-based editing. Engineered Cas variants offer diverse capabilities such as base editing, prime editing, gene insertion and gene regulation, thereby providing a panoply of tools for the scientific community. We highlight the strengths and weaknesses of current CRISPR tools, considering their efficiency, precision, specificity, reliance on cellular DNA repair mechanisms and their applications in both fundamental biology and therapeutics. Finally, we discuss ongoing clinical trials that illustrate the potential impact of CRISPR systems on human health.

Programmable RNA writing with trans-splicing.

RNA editing offers the opportunity to introduce either stable or transient modifications to nucleic acid sequence without permanent off-target effects, but installation of arbitrary edits into the transcriptome is currently infeasible. Here, we describe Programmable RNA Editing & Cleavage for Insertion, Substitution, and Erasure (PRECISE), a versatile RNA editing method for writing RNA of arbitrary length and sequence into existing pre-mRNAs via 5' or 3' trans-splicing. In trans-splicing, an exogenous template is introduced to compete with the endogenous pre-mRNA, allowing for replacement of upstream or downstream exon sequence. Using Cas7-11 cleavage of pre-mRNAs to bias towards editing outcomes, we boost the efficiency of RNA trans-splicing by 10-100 fold, achieving editing rates between 5-50% and 85% on endogenous and reporter transcripts, respectively, while maintaining high-fidelity. We demonstrate PRECISE editing across 11 distinct endogenous transcripts of widely varying expression levels, showcasing more than 50 types of edits, including all 12 possible transversions and transitions, insertions ranging from 1 to 1,863 nucleotides, and deletions. We show high efficiency replacement of exon 4 of MECP2, addressing most mutations that drive the Rett Syndrome; editing of SHANK3 transcripts, a gene involved in Autism; and replacement of exon 1 of HTT, removing the hallmark repeat expansions of Huntington's disease. Whole transcriptome sequencing reveals the high precision of PRECISE editing and lack of off-target trans-splicing activity. Furthermore, we combine payload engineering and ribozymes for protein-free, high-efficiency trans-splicing, with demonstrated efficiency in editing HTT exon 1 via AAV delivery. We show that the high activity of PRECISE editing enables editing in non-dividing neurons and patient-derived Huntington's disease fibroblasts. PRECISE editing markedly broadens the scope of genetic editing, is straightforward to deliver over existing gene editing tools like prime editing, lacks permanent off-targets, and can enable any type of genetic edit large or small, including edits not otherwise possible with existing RNA base editors, widening the spectrum of addressable diseases.

Programmable RNA-guided DNA endonucleases are widespread in eukaryotes and their viruses.

Programmable RNA-guided DNA nucleases perform numerous roles in prokaryotes, but the extent of their spread outside prokaryotes is unclear. Fanzors, the eukaryotic homolog of prokaryotic TnpB proteins, have been detected in genomes of eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. Here, we characterize Fanzors as RNA-programmable DNA endonucleases, using biochemical and cellular evidence. We found diverse Fanzors that frequently associate with various eukaryotic transposases. Reconstruction of Fanzors evolution revealed multiple radiations of RuvC-containing TnpB homologs in eukaryotes. Fanzor genes captured introns and proteins acquired nuclear localization signals, indicating extensive, long-term adaptation to functioning in eukaryotic cells. Fanzor nucleases contain a rearranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that Fanzors can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.

Programmable RNA-guided endonucleases are widespread in eukaryotes and their viruses.

TnpB proteins are RNA-guided nucleases that are broadly associated with IS200/605 family transposons in prokaryotes. TnpB homologs, named Fanzors, have been detected in genomes of some eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. We searched genomes of diverse eukaryotes and their viruses for TnpB homologs and identified numerous putative RNA-guided nucleases that are often associated with various transposases, suggesting they are encoded in mobile genetic elements. Reconstruction of the evolution of these nucleases, which we rename Horizontally-transferred Eukaryotic RNA-guided Mobile Element Systems (HERMES), revealed multiple acquisitions of TnpBs by eukaryotes and subsequent diversification. In their adaptation and spread in eukaryotes, HERMES proteins acquired nuclear localization signals, and genes captured introns, indicating extensive, long term adaptation to functioning in eukaryotic cells. Biochemical and cellular evidence show that HERMES employ non-coding RNAs encoded adjacent to the nuclease for RNA-guided cleavage of double-stranded DNA. HERMES nucleases contain a re-arranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that HERMES can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.

Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox.

CRISPR systems mediate adaptive immunity in bacteria and archaea through diverse effector mechanisms and have been repurposed for versatile applications in therapeutics and diagnostics thanks to their facile reprogramming with RNA guides. RNA-guided CRISPR-Cas targeting and interference are mediated by effectors that are either components of multisubunit complexes in class 1 systems or multidomain single-effector proteins in class 2. The compact class 2 CRISPR systems have been broadly adopted for multiple applications, especially genome editing, leading to a transformation of the molecular biology and biotechnology toolkit. The diversity of class 2 effector enzymes, initially limited to the Cas9 nuclease, was substantially expanded via computational genome and metagenome mining to include numerous variants of Cas12 and Cas13, providing substrates for the development of versatile, orthogonal molecular tools. Characterization of these diverse CRISPR effectors uncovered many new features, including distinct protospacer adjacent motifs (PAMs) that expand the targeting space, improved editing specificity, RNA rather than DNA targeting, smaller crRNAs, staggered and blunt end cuts, miniature enzymes, promiscuous RNA and DNA cleavage, etc. These unique properties enabled multiple applications, such as harnessing the promiscuous RNase activity of the type VI effector, Cas13, for supersensitive nucleic acid detection. class 1 CRISPR systems have been adopted for genome editing, as well, despite the challenge of expressing and delivering the multiprotein class 1 effectors. The rich diversity of CRISPR enzymes led to rapid maturation of the genome editing toolbox, with capabilities such as gene knockout, base editing, prime editing, gene insertion, DNA imaging, epigenetic modulation, transcriptional modulation, and RNA editing. Combined with rational design and engineering of the effector proteins and associated RNAs, the natural diversity of CRISPR and related bacterial RNA-guided systems provides a vast resource for expanding the repertoire of tools for molecular biology and biotechnology.

A transcription factor atlas of directed differentiation.

Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated candidate TFs for generation of diverse cell types, spanning all three germ layers and trophoblasts. Targeted screens with subsets of the library allowed us to create a tailored cellular disease model and integrate mRNA expression and chromatin accessibility data to identify downstream regulators. Finally, we characterized the effects of combinatorial TF overexpression by developing and validating a strategy for predicting combinations of TFs that produce target expression profiles matching reference cell types to accelerate cellular engineering efforts.

Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR.

Programmable approaches to sense and respond to the presence of specific RNAs in biological systems have broad applications in research, diagnostics, and therapeutics. Here we engineer a programmable RNA-sensing technology, reprogrammable ADAR sensors (RADARS), which harnesses RNA editing by adenosine deaminases acting on RNA (ADAR) to gate translation of a cargo protein by the presence of endogenous RNA transcripts. Introduction of a stop codon in a guide upstream of the cargo makes translation contingent on binding of an endogenous transcript to the guide, leading to ADAR editing of the stop codon and allowing translational readthrough. Through systematic sensor engineering, we achieve 277 fold improvement in sensor activation and engineer RADARS with diverse cargo proteins, including luciferases, fluorescent proteins, recombinases, and caspases, enabling detection sensitivity on endogenous transcripts expressed at levels as low as 13 transcripts per million. We show that RADARS are functional as either expressed DNA or synthetic mRNA and with either exogenous or endogenous ADAR. We apply RADARS in multiple contexts, including tracking transcriptional states, RNA-sensing-induced cell death, cell-type identification, and control of synthetic mRNA translation.

Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.

Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.

RNA-activated protein cleavage with a CRISPR-associated endopeptidase.

In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have been found in genetic association with CRISPR systems, suggesting that there may be other RNA-guided non-nucleolytic enzymes. One such gene from Desulfonema ishimotonii encodes the TPR-CHAT protease Csx29, which is associated with the CRISPR effector Cas7-11. Here, we demonstrate that this CRISPR-associated protease (CASP) exhibits programmable RNA-activated endopeptidase activity against a sigma factor inhibitor to regulate a transcriptional response. Cryo-electron microscopy of an active and substrate-bound CASP complex reveals an allosteric activation mechanism that reorganizes Csx29 catalytic residues upon target RNA binding. This work reveals an RNA-guided function in nature that can be leveraged for RNA-sensing applications in vitro and in human cells.

RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease.

The type III-E CRISPR-Cas7-11 effector binds a CRISPR RNA (crRNA) and the putative protease Csx29 and catalyzes crRNA-guided RNA cleavage. We report cryo-electron microscopy structures of the Cas7-11-crRNA-Csx29 complex with and without target RNA (tgRNA), and demonstrate that tgRNA binding induces conformational changes in Csx29. Biochemical experiments revealed tgRNA-dependent cleavage of the accessory protein Csx30 by Csx29. Reconstitution of the system in bacteria showed that Csx30 cleavage yields toxic protein fragments that cause growth arrest, which is regulated by Csx31. Csx30 binds Csx31 and the associated sigma factor RpoE (RNA polymerase, extracytoplasmic E), suggesting that Csx30-mediated RpoE inhibition modulates the cellular response to infection. We engineered the Cas7-11-Csx29-Csx30 system for programmable RNA sensing in mammalian cells. Overall, the Cas7-11-Csx29 effector is an RNA-dependent nuclease-protease.

Structure and engineering of the type III-E CRISPR-Cas7-11 effector complex.

The type III-E CRISPR-Cas effector Cas7-11, with dual RNase activities for precursor CRISPR RNA (pre-crRNA) processing and crRNA-guided target RNA cleavage, is a new platform for bacterial and mammalian RNA targeting. We report the 2.5-Å resolution cryoelectron microscopy structure of Cas7-11 in complex with a crRNA and its target RNA. Cas7-11 adopts a modular architecture comprising seven domains (Cas7.1-Cas7.4, Cas11, INS, and CTE) and four interdomain linkers. The crRNA 5' tag is recognized and processed by Cas7.1, whereas the crRNA spacer hybridizes with the target RNA. Consistent with our biochemical data, the catalytic residues for programmable cleavage in Cas7.2 and Cas7.3 neighbor the scissile phosphates before the flipped-out fourth and tenth nucleotides in the target RNA, respectively. Using structural insights, we rationally engineered a compact Cas7-11 variant (Cas7-11S) for single-vector AAV packaging for transcript knockdown in human cells, enabling in vivo Cas7-11 applications.

Programmable RNA targeting with the single-protein CRISPR effector Cas7-11.

CRISPR-Cas interference is mediated by Cas effector nucleases that are either components of multisubunit complexes-in class 1 CRISPR-Cas systems-or domains of a single protein-in class 2 systems1-3. Here we show that the subtype III-E effector Cas7-11 is a single-protein effector in the class 1 CRISPR-Cas systems originating from the fusion of a putative Cas11 domain and multiple Cas7 subunits that are derived from subtype III-D. Cas7-11 from Desulfonema ishimotonii (DiCas7-11), when expressed in Escherichia coli, has substantial RNA interference effectivity against mRNAs and bacteriophages. Similar to many class 2 effectors-and unique among class 1 systems-DiCas7-11 processes pre-CRISPR RNA into mature CRISPR RNA (crRNA) and cleaves RNA at positions defined by the target:spacer duplex, without detectable non-specific activity. We engineered Cas7-11 for RNA knockdown and editing in mammalian cells. We show that Cas7-11 has no effects on cell viability, whereas other RNA-targeting tools (such as short hairpin RNAs and Cas13) show substantial cell toxicity4,5. This study illustrates the evolution of a single-protein effector from multisubunit class 1 effector complexes, expanding our understanding of the diversity of CRISPR systems. Cas7-11 provides the basis for new programmable RNA-targeting tools that are free of collateral activity and cell toxicity.

CRISPR-based diagnostics.

The accurate and timely diagnosis of disease is a prerequisite for efficient therapeutic intervention and epidemiological surveillance. Diagnostics based on the detection of nucleic acids are among the most sensitive and specific, yet most such assays require costly equipment and trained personnel. Recent developments in diagnostic technologies, in particular those leveraging clustered regularly interspaced short palindromic repeats (CRISPR), aim to enable accurate testing at home, at the point of care and in the field. In this Review, we provide a rundown of the rapidly expanding toolbox for CRISPR-based diagnostics, in particular the various assays, preamplification strategies and readouts, and highlight their main applications in the sensing of a wide range of molecular targets relevant to human health.

A global metagenomic map of urban microbiomes and antimicrobial resistance.

We present a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities over 3 years, representing the first systematic, worldwide catalog of the urban microbial ecosystem. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance (AMR) markers, and genetic elements, including 10,928 viruses, 1,302 bacteria, 2 archaea, and 838,532 CRISPR arrays not found in reference databases. We identified 4,246 known species of urban microorganisms and a consistent set of 31 species found in 97% of samples that were distinct from human commensal organisms. Profiles of AMR genes varied widely in type and density across cities. Cities showed distinct microbial taxonomic signatures that were driven by climate and geographic differences. These results constitute a high-resolution global metagenomic atlas that enables discovery of organisms and genes, highlights potential public health and forensic applications, and provides a culture-independent view of AMR burden in cities.

Rapid SARS-CoV-2 testing in primary material based on a novel multiplex RT-LAMP assay.

BACKGROUND: Rapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose. METHODS: To avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (RT-LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK). RESULTS: Whilst specificity of standard RT-LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed RT-LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel RT-LAMP assays with SHERLOCK. CONCLUSION: In summary, this approach reveals one-step multiplexed RT-LAMP assays as a prime-option for the development of easy and cheap POC test kits.