RNA-guided Nuclease Research Articles

All-in-one adeno-associated virus delivery and genome editing by Neisseria meningitidis Cas9 in vivo

BackgroundClustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: Streptococcus pyogenes Cas9 (SpyCas9), Staphylococcus aureus Cas9 (SauCas9), and Campylobacter jejuni (CjeCas9). However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors, especially those in which Cas9 and sgRNA are combined into a single vector genome.ResultsHere, we present in vivo editing using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct protospacer adjacent motif (PAM), making it an excellent candidate for safe gene therapy applications. We find that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail-vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogram the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we deliver NmeCas9 with its sgRNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded > 35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo.ConclusionsOur findings indicate that NmeCas9 can enable the editing of disease-causing loci in vivo, expanding the targeting scope of RNA-guided nucleases.

Genome Editing in Mice Using CRISPR/Cas9 Technology.

CRISPR/Cas9 technology has revolutionized genome editing in mice, allowing for simple and rapid development of knockouts and knockins. CRISPR relies on small guide RNAs that direct the RNA-guided nuclease Cas9 to a designated genomic site using ∼20 bp of corresponding sequence. Cas9 then creates a double-strand break in the targeted loci that is either patched in an error-prone fashion to produce a frame-shift mutation, a knockout, or is repaired by recombination with donor DNA containing homology arms, a knockin. This protocol covers the techniques needed to rapidly generate knockout and knockin mice with CRISPR via microinjection of Cas9, the guide RNA, and possible donor DNA into the mouse zygote. © 2018 by John Wiley & Sons, Inc.

Crispr Cas System in Plant Genome Editing a New Opportunity in Agriculture to Boost Crop Yield

Clustered regularly interspaced short palindromic repeats CRISPR/Cas9 technology evolved from a type II bacterial immune system develop in 2013 This system employs RNA-guided nuclease, CRISPR associated (Cas9) to induce double-strand breaks. The Cas9-mediated breaks are repaired by cellular DNA repair mechanisms and mediate gene/genome modifications. The system has the ability to detect specific sequences of letters within the genetic code and to cut DNA at a specific point. Simultaneously with other sequence-specific nucleases, CRISPR/ Cas9 have already breach the boundaries and made genetic engineering much more versatile, efficient and easy also it has been reported to have increased rice grain yield up to 25-30 %, and increased tomato fruits size, branching architecture, and overall plant shape. CRISPR/ Cas also mediated virus resistance in many agricultural crops. In this article, we reviewed the history of the CRISPR/Cas9 system invention and its genome-editing mechanism. We also described the most recent innovation of the CRISPR/Cas9 technology, particularly the broad applications of modified Cas9 variants, and discuss the potential of this system for targeted genome editing and modification for crop improvement.
 Abbreviations: CRISPR, clustered regularly interspaced short palindromic repeats; Cas, CRISPR associated; crRNA, CRISPR RNA; tracrRNA, trans-activating crRNA; PAM, protospacer adjacent motif; sgRNA, single guide RNA; gRNA, guide RNA; ssODN, single-stranded DNA oligonucleotide; DSB, double-strand break; NHEJ, non-homologous end joining; HDR, homology directed repair, CRISPRi ,CRISPR interference

Targeted Nucleotide Editing Technologies for Microbial Metabolic Engineering.

Since the emergence of programmable RNA-guided nucleases based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems, genome editing technologies have become a simplified and versatile tool for genome editing in various organisms and cell types. Although genome editing enables efficient genome manipulations, such as gene disruptions, gene knockins, and chromosomal translocations via DNA double-strand break (DSB) repair in eukaryotes, DSBs induced by the CRISPR/Cas system are lethal or severely toxic to many microorganisms. Therefore, in many prokaryotes, including industrially useful microbes, the CRISPR/Cas system is often used as a negative selection component in combination with recombineering or other related strategies. Novel and revolutionary technologies have been recently developed to re-write targeted nucleotides (C:G to T:A and A:T to G:C) without DSBs and donor DNA templates. These technologies rely on the recruitment of deaminases at specific target loci using the nuclease-deficient CRISPR/Cas system. Here, the authors review and compare CRISPR-based genome editing, current base editing platforms and their spectra. The authors discuss how these technologies can be applied in various aspects of microbial metabolic engineering to overcome barriers to cellular regulation in prokaryotes.

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity.

The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S.pyogenes (Spy), S.aureus (Sau), and C.jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.

History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology.

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are well-known acquired immunity systems that are widespread in archaea and bacteria. The RNA-guided nucleases from CRISPR-Cas systems are currently regarded as the most reliable tools for genome editing and engineering. The first hint of their existence came in 1987, when an unusual repetitive DNA sequence, which subsequently was defined as a CRISPR, was discovered in the Escherichia coli genome during an analysis of genes involved in phosphate metabolism. Similar sequence patterns were then reported in a range of other bacteria as well as in halophilic archaea, suggesting an important role for such evolutionarily conserved clusters of repeated sequences. A critical step toward functional characterization of the CRISPR-Cas systems was the recognition of a link between CRISPRs and the associated Cas proteins, which were initially hypothesized to be involved in DNA repair in hyperthermophilic archaea. Comparative genomics, structural biology, and advanced biochemistry could then work hand in hand, not only culminating in the explosion of genome editing tools based on CRISPR-Cas9 and other class II CRISPR-Cas systems but also providing insights into the origin and evolution of this system from mobile genetic elements denoted casposons. To celebrate the 30th anniversary of the discovery of CRISPR, this minireview briefly discusses the fascinating history of CRISPR-Cas systems, from the original observation of an enigmatic sequence in E. coli to genome editing in humans.

Engineering Point Mutant and Epitope-Tagged Alleles in Mice Using Cas9 RNA-Guided Nuclease.

Mice carrying patient-associated point mutations are powerful tools to define the causality of single-nucleotide variants to disease states. Epitope tags enable immuno-based studies of genes for which no antibodies are available. These alleles enable detailed and precise developmental, mechanistic, and translational research. The first step in generating these alleles is to identify within the target sequence-the orthologous sequence for point mutations or the N or C terminus for epitope tags-appropriate Cas9 protospacer sequences. Subsequent steps include design and acquisition of a single-stranded oligonucleotide repair template, synthesis of a single guide RNA (sgRNA), collection of zygotes, and microinjection or electroporation of zygotes with Cas9 mRNA or protein, sgRNA, and repair template followed by screening of born mice for the presence of the desired sequence change. Quality control of mouse lines includes screening for random or multicopy insertions of the repair template and, depending on sgRNA sequence, off-target mutations introduced by Cas9. © 2018 by John Wiley & Sons, Inc.

A detailed cell-free transcription-translation-based assay to decipher CRISPR protospacer-adjacent motifs.

The RNA-guided nucleases derived from the CRISPR-Cas systems in bacteria and archaea have found numerous applications in biotechnology, including genome editing, imaging, and gene regulation. However, the discovery of novel Cas nucleases has outpaced their characterization and subsequent exploitation. A key step in characterizing Cas nucleases is determining which protospacer-adjacent motif (PAM) sequences they recognize. Here, we report advances to an in vitro method based on an E. coli cell-free transcription-translation system (TXTL) to rapidly elucidate PAMs recognized by Cas nucleases. The method obviates the need for cloning Cas nucleases or gRNAs, does not require the purification of protein or RNA, and can be performed in less than a day. To advance our previously published method, we incorporated an internal GFP cleavage control to assess the extent of library cleavage as well as Sanger sequencing of the cleaved library to assess PAM depletion prior to next-generation sequencing. We also detail the methods needed to construct all relevant DNA constructs, and how to troubleshoot the assay. We finally demonstrate the technique by determining PAM sequences recognized by the Neisseria meningitidis Cas9, revealing subtle sequence requirements of this highly specific PAM. The overall method offers a rapid means to identify PAMs recognized by diverse CRISPR nucleases, with the potential to greatly accelerate our ability to characterize and harness novel CRISPR nucleases across their many uses.

Enhanced Proofreading Governs CRISPR-Cas9 Targeting Accuracy

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule Förster resonance energy transfer (smFRET), we identified an intermediate state of Streptococcus pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. We showed that the high-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) Cas9 variants are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

A human iPS cell-based model of medulloblastoma demonstrates co-operativity between SHH signalling and mutation in an epigenetic modifier

Medulloblastoma (MB), the most common malignant pediatric brain tumor, is comprised of four subgroups (WNT, SHH, Group 3, Group 4). While recurrent single nucleotide variations is rare in MB, mutation of epigenetic modifiers is frequent, suggesting a mechanism for tumorigenesis. Genetically engineered mouse models (GEMMs) recapitulate some aspects of MB, but fail to replicate the epigenetics characteristic of human MB. Therefore, we aimed to develop the first human stem cell based model of MB using iPSC derived from Gorlin syndrome (GS), a disease that predisposes patients to SHH MB due to heterozygosity for PTCH1. We differentiated iPSC from healthy (WT) or GS patients toward hindbrain neuroepithelial stem (NES) cells, a presumed cell of origin of MB. We then electroporated the NES cells with RNA-guided FokI Nucleases (RFN, CRISPR/dCas9-FokI) targeting GSE1 (formerly KIAA0182), a gene encoding an epigenetic modifier and co-mutated with PTCH1 in MB. These NSC were implanted orthotopically into the hindbrains of mice. By 92 days post-implantation, 80% of mice with GS NSC mutant for GSE1 developed tumors with histology consistent with MB. In contrast, GS NSC with wild-type GSE1 developed the first tumor at longer latency (> 140 days) while WT NSC have not generated tumors (> 300 days). Our human iPSC-based model of MB demonstrates a cooperative effect between SHH signaling and a mutation in an epigenetic modifier. We are currently investigating similarities and differences between our human MB with GEMMs and elucidating the role of epigenetic alterations in MB tumorigenesis.

A beginner's guide to gene editing.

What is the topic of this review? This review summarizes the development of gene editing from early proof-of-concept studies in the 1980s to contemporary programmable and RNA-guided nucleases, which enable rapid and precise alteration of DNA sequences of almost any living cell. What advances does it highlight? With an average of one clustered regularly interspaced short palindromic repeat (CRISPR) Cas9 paper published every 4h in 2017, this review cannot highlight all new developments, but a number of key improvements, including increases in efficiency, a range of new options to reduce off-target effects and plans for CRISPR to enter clinical trials in 2018, are discussed. Genome editing enables precise changes to be made in the genome of living cells. The technique was originally developed in the 1980s but largely limited to use in mice. The discovery that a targeted double-stranded break at a unique site in the genome, close to the site to be changed, could substantially increase the efficiency of editing raised the possibility of using the technique in a broader range of animal models and, potentially, human cells. But the challenge was to identify reagents that could create targeted breaks at a unique genomic location with minimal off-target effects. In 2005, the demonstration that programmable zinc finger nucleases (ZFNs) could perform this task led to a number of proof-of-concept studies, but a limitation was the ease with which effective ZFNs could be produced. In 2009, the development of TAL effector nucleases (TALENs) increased the specificity of gene editing and the ease of design and production. However, it was not until 2013 and the development of the clustered regularly interspaced short palindromic repeat (CRISPR) Cas9/guide RNA that gene editing became a research tool that any laboratory could use.

Genome engineering technologies for targeted genetic modification in plants

Well-established targeted technologies to engineer genomes such as zinc-finger nuclease-based editing (ZFN), transcription activator-like effector nuclease-based editing (TALEN), and clustered regularly interspaced short palindromic repeats and associated protein system-based editing (CRISPR/Cas) are proving to advance basic and applied research in numerous plant species. Compared with systems using ZFNs and TALENs, the most recently developed CRISPR/Cas system is more efficient due to its use of an RNA-guided nuclease to generate double-strand DNA breaks. To accelerate the applications of these technologies, we provide here a detailed overview of these systems, highlight the strengths and weaknesses of each, summarize research advances made with these technologies in model and crop plants, and discuss their applications in plant functional genomics. Such targeted approaches for genetically modifying plants will benefit agricultural production in the future.

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells.

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

CRISPR-Cas9-Mediated Genome Editing and Transcriptional Control in Yarrowia lipolytica.

The discovery and adaptation of RNA-guided nucleases has resulted in the rapid development of efficient, scalable, and easily accessible synthetic biology tools for targeted genome editing and transcriptional control. In these systems, for example CRISPR-Cas9 from Streptococcus pyogenes, a protein with nuclease activity is targeted to a specific nucleotide sequence by a short RNA molecule, whereupon binding it cleaves the targeted nucleotide strand. To extend this genome-editing ability to the industrially important oleaginous yeast Yarrowia lipolytica, we developed a set of easily usable and effective CRISPR-Cas9 episomal vectors. In this protocols chapter, we first present a method by which arbitrary protein-coding genes can be disrupted via indel formation after CRISPR-Cas9 targeting. A second method demonstrates how the same CRISPR-Cas9 system can be used to induce markerless gene cassette integration into the genome by inducing homologous recombination after DNA cleavage by Cas9. Finally, we describe how a catalytically inactive form of Cas9 fused to a transcriptional repressor can be used to control transcription of native genes in Y. lipolytica. The CRISPR-Cas9 tools and strategies described here greatly increase the types of genome editing and transcriptional control that can be achieved in Y. lipolytica, and promise to facilitate more advanced engineering of this important oleaginous host.

In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting

Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach.

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1–4. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown5–9. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

Progress and prospects in plant genome editing.

The emergence of sequence-specific nucleases that enable genome editing is revolutionizing basic and applied biology. Since the introduction of CRISPR-Cas9, genome editing has become widely used in transformable plants for characterizing gene function and improving traits, mainly by inducing mutations through non-homologous end joining of double-stranded breaks generated by CRISPR-Cas9. However, it would be highly desirable to perform precision gene editing in plants, especially in transformation-recalcitrant species. Recently developed Cas9 variants, novel RNA-guided nucleases and base-editing systems, and DNA-free CRISPR-Cas9 delivery methods now provide great opportunities for plant genome engineering. In this Review Article, we describe the current status of plant genome editing, focusing on newly developed genome editing tools and methods and their potential applications in plants. We also discuss the specific challenges facing plant genome editing, and future prospects.

The Discovery, Mechanisms, and Evolutionary Impact of Anti-CRISPRs.

Bacteria and archaea use CRISPR-Cas adaptive immune systems to defend themselves from infection by bacteriophages (phages). These RNA-guided nucleases are powerful weapons in the fight against foreign DNA, such as phages and plasmids, as well as a revolutionary gene editing tool. Phages are not passive bystanders in their interactions with CRISPR-Cas systems, however; recent discoveries have described phage genes that inhibit CRISPR-Cas function. More than 20 protein families, previously of unknown function, have been ascribed anti-CRISPR function. Here, we discuss how these CRISPR-Cas inhibitors were discovered and their modes of action were elucidated. We also consider the potential impact of anti-CRISPRs on bacterial and phage evolution. Finally, we speculate about the future of this field.

CRISPR/Cas9-Mediated Knockin Application in Cell Therapy: A Non-viral Procedure for Bystander Treatment of Glioma in Mice.

The use of non-viral procedures, together with CRISPR/Cas9 genome-editing technology, allows the insertion of single-copy therapeutic genes at pre-determined genomic sites, overcoming safety limitations resulting from random gene insertions of viral vectors with potential for genome damage. In this study, we demonstrate that combination of non-viral gene delivery and CRISPR/Cas9-mediated knockin via homology-directed repair can replace the use of viral vectors for the generation of genetically modified therapeutic cells. We custom-modified human adipose mesenchymal stem cells (hAMSCs), using electroporation as a transfection method and CRISPR/Cas9-mediated knockin for the introduction and stable expression of a 3 kb DNA fragment including the eGFP (selectable marker) and a variant of the herpes simplex virus 1 thymidine kinase genes (therapeutic gene), under the control of the human elongation factor 1 alpha promoter in exon 5 of the endogenous thymidine kinase 2 gene. Using a U87 glioma model in SCID mice, we show that the therapeutic capacity of the new CRISPR/Cas9-engineered hAMSCs is equivalent to that of therapeutic hAMSCs generated by introduction of the same therapeutic gene by transduction with a lentiviral vector previously published by our group. This strategy should be of general use to other applications requiring genetic modification of therapeutic cells.