CRISPR-Cas Nucleases Research Articles

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.

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.

Engineering of Bacteriophage T4 Genome Using CRISPR-Cas9.

Bacteriophages likely constitute the largest biomass on Earth. However, very few phage genomes have been well-characterized, the tailed phage T4 genome being one of them. Even in T4, much of the genome remained uncharacterized. The classical genetic strategies are tedious, compounded by genome modifications such as cytosine hydroxylmethylation and glucosylation which makes T4 DNA resistant to most restriction endonucleases. Here, using the type-II CRISPR-Cas9 system, we report the editing of both modified (ghm-Cytosine) and unmodified (Cytosine) T4 genomes. The modified genome, however, is less susceptible to Cas9 nuclease attack when compared to the unmodified genome. The efficiency of restriction of modified phage infection varied greatly in a spacer-dependent manner, which explains some of the previous contradictory results. We developed a genome editing strategy by codelivering into E. coli a CRISPR-Cas9 plasmid and a donor plasmid containing the desired mutation(s). Single and multiple point mutations, insertions and deletions were introduced into both modified and unmodified genomes. As short as 50-bp homologous flanking arms were sufficient to generate recombinants that can be selected under the pressure of CRISPR-Cas9 nuclease. A 294-bp deletion in RNA ligase gene rnlB produced viable plaques, demonstrating the usefulness of this editing strategy to determine the essentiality of a given gene. These results provide the first demonstration of phage T4 genome editing that might be extended to other phage genomes in nature to create useful recombinants for phage therapy applications.

GUIDEseq: a bioconductor package to analyze GUIDE-Seq datasets for CRISPR-Cas nucleases

BackgroundGenome editing technologies developed around the CRISPR-Cas9 nuclease system have facilitated the investigation of a broad range of biological questions. These nucleases also hold tremendous promise for treating a variety of genetic disorders. In the context of their therapeutic application, it is important to identify the spectrum of genomic sequences that are cleaved by a candidate nuclease when programmed with a particular guide RNA, as well as the cleavage efficiency of these sites. Powerful new experimental approaches, such as GUIDE-seq, facilitate the sensitive, unbiased genome-wide detection of nuclease cleavage sites within the genome. Flexible bioinformatics analysis tools for processing GUIDE-seq data are needed.ResultsHere, we describe an open source, open development software suite, GUIDEseq, for GUIDE-seq data analysis and annotation as a Bioconductor package in R. The GUIDEseq package provides a flexible platform with more than 60 adjustable parameters for the analysis of datasets associated with custom nuclease applications. These parameters allow data analysis to be tailored to different nuclease platforms with different length and complexity in their guide and PAM recognition sequences or their DNA cleavage position. They also enable users to customize sequence aggregation criteria, and vary peak calling thresholds that can influence the number of potential off-target sites recovered. GUIDEseq also annotates potential off-target sites that overlap with genes based on genome annotation information, as these may be the most important off-target sites for further characterization. In addition, GUIDEseq enables the comparison and visualization of off-target site overlap between different datasets for a rapid comparison of different nuclease configurations or experimental conditions. For each identified off-target, the GUIDEseq package outputs mapped GUIDE-Seq read count as well as cleavage score from a user specified off-target cleavage score prediction algorithm permitting the identification of genomic sequences with unexpected cleavage activity.ConclusionThe GUIDEseq package enables analysis of GUIDE-data from various nuclease platforms for any species with a defined genomic sequence. This software package has been used successfully to analyze several GUIDE-seq datasets. The software, source code and documentation are freely available at http://www.bioconductor.org/packages/release/bioc/html/GUIDEseq.html.

CRISPR-Cas9 Mediated DNA Unwinding Detected Using Site-Directed Spin Labeling.

The RNA-guided CRISPR-Cas9 nuclease has revolutionized genome engineering, yet its mechanism for DNA target selection is not fully understood. A crucial step in Cas9 target recognition involves unwinding of the DNA duplex to form a three-stranded R-loop structure. Work reported here demonstrates direct detection of Cas9-mediated DNA unwinding by a combination of site-directed spin labeling and molecular dynamics simulations. The results support a model in which the unwound nontarget strand is stabilized by a positively charged patch located between the two nuclease domains of Cas9 and reveal uneven increases in flexibility along the unwound nontarget strand upon scissions of the DNA backbone. This work establishes the synergistic combination of spin-labeling and molecular dynamics to directly monitor Cas9-mediated DNA conformational changes and yields information on the target DNA in different stages of Cas9 function, thus advancing mechanistic understanding of CRISPR-Cas9 and aiding future technological development.

CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets.

Sensitive detection of off-target effects is important for translating CRISPR-Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe CIRCLE-seq (Circularization for In vitro Reporting of CLeavage Effects by sequencing), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR-Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequence. Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific SNPs, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides the most accessible, rapid and comprehensive method for identifying genome-wide off-target mutations of CRISPR-Cas9 described to date.

The new generation tool for CRISPR genome editing: CRISPR/Cpf1

Almost all archaea and many bacteria achieve adaptive immunity through a diverse set of CRISPR-Cas systems. From 2012, Cas9 has been harnessed by thousands of laboratories for genome editing applications in a variety of experimental model systems. Cas9 is driving innovative applications from basic biology to biotechnology and medicine. However, numerous challenges still remain, such as limited targeting range, toxicity, and potential for off-target mutagenesis. Cpf1 represents a class 2/type V CRISPR RNA-guided endonuclease that is distinct from the type II CRISPR Cas9 nuclease. Future exploration of strategies using Cpf1 is expected to bring solutions for some of the biggest challenges facing genome editing. Here, we review the development and applications of Cpf1 for a variety of research or applications, and highlight challenges as well as future directions.

Genom-Editierung — neue Wege im klinischen Alltag

Programmable nucleases, such as zinc finger nucleases, TALE nucleases, and CRISPR-Cas nucleases, allow for the introduction of precise modifications in the human genome. As a consequence, targeted genome editing has heralded a new area for gene therapy and the treatment of acquired and inherited disorders. Here, we describe the concept of designer nuclease- mediated genome engineering, discuss risks and potential limitations of its usage, highlight some of the achieved milestones, and review the most prominent studies that have made it from the laboratory to the clinic.

Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration.

RNA-guided genome surgery using CRISPR-Cas9 nucleases has shown promise for the treatment of diverse genetic diseases. Yet, the potential of such nucleases for therapeutic applications in nongenetic diseases is largely unexplored. Here, we focus on age-related macular degeneration (AMD), a leading cause of blindness in adults, which is associated with retinal overexpression of, rather than mutations in, the VEGFA gene. Subretinal injection of preassembled, Vegfa gene–specific Cas9 ribonucleoproteins (RNPs) into the adult mouse eye gave rise to mutagenesis at the target site in the retinal pigment epithelium. Furthermore, Cas9 RNPs effectively reduced the area of laser-induced choroidal neovascularization (CNV) in a mouse model of AMD. Genome-wide profiling of Cas9 off-target effects via Digenome-seq showed that off-target mutations were rarely induced in the human genome. Because Cas9 RNPs can function immediately after in vivo delivery and are rapidly degraded by endogenous proteases, their activities are unlikely to be hampered by antibody- and cell-mediated adaptive immune systems. Our results demonstrate that in vivo genome editing with Cas9 RNPs has the potential for the local treatment for nongenetic degenerative diseases, expanding the scope of RNA-guided genome surgery to a new dimension.

Therapeutic genome editing with engineered nucleases.

Targeted genome editing with designer nucleases, such as zinc finger nucleases, TALE nucleases, and CRISPR-Cas nucleases, has heralded a new era in gene therapy. Genetic disorders, which have not been amenable to conventional gene-addition-type gene therapy approaches, such as disorders with dominant inheritance or diseases caused by mutations in tightly regulated genes, can now be treated by precise genome surgery. Moreover, engineered nucleases enable novel genetic interventions to fight infectious diseases or to improve cancer immunotherapies. Here, we review the development of the different classes of programmable nucleases, discuss the challenges and improvements in translating gene editing into clinical use, and give an outlook on what applications can expect to enter the clinic in the near future.

Rapid Construction of Multiplexed CRISPR-Cas9 Systems for Plant Genome Editing.

Multiplex CRISPR-Cas9 nuclease mediated genome editing is an efficient method for disrupting gene function in plants. Use of CRISPR-Cas9 has escalated rapidly in recent years and is expected to become routine practice in molecular biology and related fields of research. Due to the relatively novel and widespread adoption of this technology, first-time users may not have regular access to experienced guidance or technical support from peers or mentors. Here, we offer guidance and technical support in the form of a detailed and tested protocol for simultaneous targeting of three separate loci on the TRANSPARENT TESTA 4 (TT4) gene in Arabidopsis thaliana using multiplex CRISPR-Cas9. Although we target multiple loci on a single gene in Arabidopsis, the same approach can be used to target multiple genes or alleles in other plant species as well. We recommend the use of a molecular toolkit to streamline the process and make recommendations for this type of approach. The protocol starts with an overview of the reagents and covers designing of gRNAs and assembly of components into a final T-DNA delivery molecule through Golden Gate cloning and Multisite Gateway LR recombination.

Gene therapy: Gene-editing therapy for neurological disease.

Guide RNA-mediated CRISPR–Cas nucleases are a powerful technology for the engineering of mammalian genomes. CRISPR–Cas9-dependent editing of mutated genes that cause Huntington disease and fragile X syndrome was recently achieved in cell-based models, heralding the first step towards developing this technology into viable therapeutics for neurological diseases.

Cas-analyzer: an online tool for assessing genome editing results using NGS data.

SummaryGenome editing with programmable nucleases has been widely adopted in research and medicine. Next generation sequencing (NGS) platforms are now widely used for measuring the frequencies of mutations induced by CRISPR-Cas9 and other programmable nucleases. Here, we present an online tool, Cas-Analyzer, a JavaScript-based implementation for NGS data analysis. Because Cas-Analyzer is completely used at a client-side web browser on-the-fly, there is no need to upload very large NGS datasets to a server, a time-consuming step in genome editing analysis. Currently, Cas-Analyzer supports various programmable nucleases, including single nucleases and paired nucleases.Availability and ImplementationFree access at http://www.rgenome.net/cas-analyzer/.Supplementary information Supplementary data are available at Bioinformatics online.

Targeting safety in the clinic for precise genome editing using CRISPR: a genotoxicologist's perspective.

Targeting safety in the clinic for precise genome editing using CRISPR: a genotoxicologist's perspective.

Novel Hypomorphic Alleles of the Mouse Tyrosinase Gene Induced by CRISPR-Cas9 Nucleases Cause Non-Albino Pigmentation Phenotypes.

Tyrosinase is a key enzyme in melanin biosynthesis. Mutations in the gene encoding tyrosinase (Tyr) cause oculocutaneous albinism (OCA1) in humans. Alleles of the Tyr gene have been useful in studying pigment biology and coat color formation. Over 100 different Tyr alleles have been reported in mice, of which ≈24% are spontaneous mutations, ≈60% are radiation-induced, and the remaining alleles were obtained by chemical mutagenesis and gene targeting. Therefore, most mutations were random and could not be predicted a priori. Using the CRISPR-Cas9 system, we targeted two distinct regions of exon 1 to induce pigmentation changes and used an in vivo visual phenotype along with heteroduplex mobility assays (HMA) as readouts of CRISPR-Cas9 activity. Most of the mutant alleles result in complete loss of tyrosinase activity leading to an albino phenotype. In this study, we describe two novel in-frame deletion alleles of Tyr, dhoosara (Sanskrit for gray) and chandana (Sanskrit for sandalwood). These alleles are hypomorphic and show lighter pigmentation phenotypes of the body and eyes. This study demonstrates the utility of CRISPR-Cas9 system in generating domain-specific in-frame deletions and helps gain further insights into structure-function of Tyr gene.

731. High-Fidelity CRISPR-Cas9 Nucleases with No Detectable Genome-Wide Off-Target Effects

CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations at genomic locations that resemble the intended target. These so-called off-target effects can confound research applications and are important considerations for potential therapeutic use. Existing strategies for reducing genome-wide off-targets of the broadly used Streptococcus pyogenes Cas9 (SpCas9) have thus far proven to be imperfect by possessing only partial efficacy and/or other limitations that constrain their use. Here we describe a high-fidelity variant of SpCas9, called SpCas9-HF1, that contains alterations in the amino acid sequence designed to reduce non-specific contacts to the target strand DNA. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of the 37 single-guide RNAs (sgRNAs) tested in human cells. Strikingly, with eight different sgRNAs targeted to standard non-repetitive sequences in human cells, SpCas9-HF1 rendered all or nearly all off-target events imperceptible by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-targets induced by SpCas9-HF1 were not detected. With its exceptional precision, SpCas9-HF1 provides an important and easily employed alternative to wild-type SpCas9 that can eliminate off-target effects when using CRISPR-Cas9 for research and therapeutic applications. Our findings also suggest a general strategy for improving or optimizing the genome-wide specificities of other Cas9 orthologues and engineered variants.

Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases.

CRISPR-Cas9 RNA-guided nucleases are a transformative technology for biology, genetics and medicine owing to the simplicity with which they can be programmed to cleave specific DNA target sites in living cells and organisms. However, to translate these powerful molecular tools into safe, effective clinical applications, it is of crucial importance to carefully define and improve their genome-wide specificities. Here, we outline our state-of-the-art understanding of target DNA recognition and cleavage by CRISPR-Cas9 nucleases, methods to determine and improve their specificities, and key considerations for how to evaluate and reduce off-target effects for research and therapeutic applications.

Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases

Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing.

Genome editing: The domestication of Cas9.

The enzyme Cas9 is used in genome editing to cut selected DNA sequences, but it also creates breaks at off-target sites. Protein engineering has now been used to make Cas9 enzymes that have minimal off-target effects. See Article p.490 The CRISPR–Cas9 nucleases now widely used in gene editing can be readily customized, but can also induce substantial genome-wide off-target mutations at sequences that resemble the on-target site. Keith Joung and colleagues report a high-fidelity variant of Cas9 from Streptococcus pyogenes that shows on-target activities comparable to the wild-type enzyme, but with off-target events that are undetectable by genome-wide break capture and targeted sequencing methods.

Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq

We present multiplex Digenome-seq to profile genome-wide specificities of up to 11 CRISPR-Cas9 nucleases simultaneously, saving time and reducing cost. Cell-free human genomic DNA was digested using multiple sgRNAs combined with the Cas9 protein and then subjected to whole-genome sequencing. In vitro cleavage patterns, characteristic of on- and off-target sites, were computationally identified across the genome using a new DNA cleavage scoring system. We found that many false-positive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribed from a plasmid template. Multiplex Digenome-seq captured many bona fide off-target sites, missed by other genome-wide methods, at which indels were induced at frequencies <0.1%. After analyzing 964 sites cleaved in vitro by these sgRNAs and measuring indel frequencies at hundreds of off-target sites in cells, we propose a guideline for the choice of target sites for minimizing CRISPR-Cas9 off-target effects in the human genome.