Clustered Regularly Interspaced Short Palindromic Repeats System Research Articles

CRISPR-induced indels and base editing using the Staphylococcus aureus Cas9 in potato.

Genome editing is now widely used in plant science for both basic research and molecular crop breeding. The clustered regularly interspaced short palindromic repeats (CRISPR) technology, through its precision, high efficiency and versatility, allows for editing of many sites in plant genomes. This system has been highly successful to produce knock-out mutants through the introduction of frameshift mutations due to error-prone repair pathways. Nevertheless, recent new CRISPR-based technologies such as base editing and prime editing can generate precise and on demand nucleotide conversion, allowing for fine-tuning of protein function and generating gain-of-function mutants. However, genome editing through CRISPR systems still have some drawbacks and limitations, such as the PAM restriction and the need for more diversity in CRISPR tools to mediate different simultaneous catalytic activities. In this study, we successfully used the CRISPR-Cas9 system from Staphylococcus aureus (SaCas9) for the introduction of frameshift mutations in the tetraploid genome of the cultivated potato (Solanum tuberosum). We also developed a S. aureus-cytosine base editor that mediate nucleotide conversions, allowing for precise modification of specific residues or regulatory elements in potato. Our proof-of-concept in potato expand the plant dicot CRISPR toolbox for biotechnology and precision breeding applications.

The CARF Protein MM_0565 Affects Transcription of the Casposon-Encoded cas1-solo Gene in Methanosarcina mazei Gö1.

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci are found in bacterial and archaeal genomes where they provide the molecular machinery for acquisition of immunity against foreign DNA. In addition to the cas genes fundamentally required for CRISPR activity, a second class of genes is associated with the CRISPR loci, of which many have no reported function in CRISPR-mediated immunity. Here, we characterize MM_0565 associated to the type I-B CRISPR-locus of Methanosarcina mazei Gö1. We show that purified MM_0565 composed of a CRISPR-Cas Associated Rossmann Fold (CARF) and a winged helix-turn-helix domain forms a dimer in solution; in vivo, the dimeric MM_0565 is strongly stabilized under high salt stress. While direct effects on CRISPR-Cas transcription were not detected by genetic approaches, specific binding of MM_0565 to the leader region of both CRISPR-Cas systems was observed by microscale thermophoresis and electromobility shift assays. Moreover, overexpression of MM_0565 strongly induced transcription of the cas1-solo gene located in the recently reported casposon, the gene product of which shows high similarity to classical Cas1 proteins. Based on our findings, and taking the absence of the expressed CRISPR locus-encoded Cas1 protein into account, we hypothesize that MM_0565 might modulate the activity of the CRISPR systems on different levels.

Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing.

Gene editing that makes target gene modification in the genome by deletion or addition has revolutionized the era of biomedicine. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 emerged as a substantial tool due to its simplicity in use, less cost and extraordinary efficiency than the conventional gene-editing tools, including zinc finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs). However, potential off-target activities are crucial shortcomings in the CRISPR system. Numerous types of approaches have been developed to reduce off-target effects. Here, we review several latest approaches to reduce the off-target effects, including biased or unbiased off-target detection, cytosine or adenine base editors, prime editing, dCas9, Cas9 paired nickase, ribonucleoprotein (RNP) delivery and truncated gRNAs. This review article provides extensive information to cautiously interpret off-target effects to assist the basic and clinical applications in biomedicine.

Applications of CRISPR for musculoskeletal research.

The ability to edit DNA at the nucleotide level using clustered regularly interspaced short palindromic repeats (CRISPR) systems is a relatively new investigative tool that is revolutionizing the analysis of many aspects of human health and disease, including orthopaedic disease. CRISPR, adapted for mammalian cell genome editing from a bacterial defence system, has been shown to be a flexible, programmable, scalable, and easy-to-use gene editing tool. Recent improvements increase the functionality of CRISPR through the engineering of specific elements of CRISPR systems, the discovery of new, naturally occurring CRISPR molecules, and modifications that take CRISPR beyond gene editing to the regulation of gene transcription and the manipulation of RNA. Here, the basics of CRISPR genome editing will be reviewed, including a description of how it has transformed some aspects of molecular musculoskeletal research, and will conclude by speculating what the future holds for the use of CRISPR-related treatments and therapies in clinical orthopaedic practice.Cite this article: Bone Joint Res 2020;9(7):351–359.

Development and Application of CRISPR/Cas in Microbial Biotechnology.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has been rapidly developed as versatile genomic engineering tools with high efficiency, accuracy and flexibility, and has revolutionized traditional methods for applications in microbial biotechnology. Here, key points of building reliable CRISPR/Cas system for genome engineering are discussed, including the Cas protein, the guide RNA and the donor DNA. Following an overview of various CRISPR/Cas tools for genome engineering, including gene activation, gene interference, orthogonal CRISPR systems and precise single base editing, we highlighted the application of CRISPR/Cas toolbox for multiplexed engineering and high throughput screening. We then summarize recent applications of CRISPR/Cas systems in metabolic engineering toward production of chemicals and natural compounds, and end with perspectives of future advancements.

CRISPR Genome Editing Technology and its Application in Genetic Diseases: A Review.

Gene therapy has been a long lasting goal for scientists, and there are many optimal methods and tools to correct disease-causing mutations in humans. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been progressively adopted for the assessment a treatment of human diseases, including thalassemia, Parkinson's disease, cystic fibrosis, glaucoma, Huntington's disease, and Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS). CRISPR sequences belong to the bacterial immune system, which includes the nuclease Cas enzyme and an RNA sequence. The RNA sequence is unique and pathogen-specific, and identifies and binds to the DNA of invasive viruses, allowing the nuclease Cas enzyme to cut the identified DNA and destroy the invasive viruses. This feature provides the possibility to edit mutations in the DNA sequence of live cells by replacing a specific targeted RNA sequence with the RNA sequence in the CRISPR system. Previous studies have reported the improvement steps in confrontation with human diseases caused by single-nucleotide mutations using this system. In this review, we first introduce CRISPR and its functions and then elaborate on the use of CRISPR in the treatment of human diseases.

Progress and Challenges in the Improvement of Ornamental Plants by Genome Editing.

Biotechnological approaches have been used to modify the floral color, size, and fragrance of ornamental plants, as well as to increase disease resistance and vase life. Together with the advancement of whole genome sequencing technologies, new plant breeding techniques have rapidly emerged in recent years. Compared to the early versions of gene editing tools, such as meganucleases (MNs), zinc fingers (ZFNs), and transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeat (CRISPR) is capable of altering a genome more efficiently and with higher accuracy. Most recently, new CRISPR systems, including base editors and prime editors, confer reduced off-target activity with improved DNA specificity and an expanded targeting scope. However, there are still controversial issues worldwide for the recognition of genome-edited plants, including whether genome-edited plants are genetically modified organisms and require a safety evaluation process. In the current review, we briefly summarize the current progress in gene editing systems and also introduce successful/representative cases of the CRISPR system application for the improvement of ornamental plants with desirable traits. Furthermore, potential challenges and future prospects in the use of genome-editing tools for ornamental plants are also discussed.

The Manipulation of RNA-Guided Nucleic Acid Cleavage with Ninhydrin Chemistry.

CRISPR (clustered regularly interspaced short palindromic repeats) systems have been established as valuable genome‐editing tools. Controlling CRISPR systems has high biological significance and this field has garnered intense interest. There is a considerable need for simple approaches with no need for protein engineering. The CRISPR systems usually require a guide RNA (gRNA) moiety to recruit and direct the nuclease complexes. In this respect, the ninhydrin (1,2,3‐indantrione monohydrate) seems to have considerable potential, as yet unexploited, for modifying gRNA. In this study, ninhydrin chemistry is explored for reversible postsynthetic modification of gRNA molecules. It is further shown that ninhydrin chemistry is efficient in modulating two important CRISPR systems. Thus, ninhydrin chemistry exhibits potential applications in future chemical biology studies.

Avoidance of Self during CRISPR Immunization.

The battle between microbes and their viruses is ancient and ongoing. Clustered regularly interspaced short palindromic repeat (CRISPR) immunity, the first and, to date, only form of adaptive immunity found in prokaryotes, represents a flexible mechanism to recall past infections while also adapting to a changing pathogenic environment. Critical to the role of CRISPR as an adaptive immune mechanism is its capacity for self versus non-self recognition when acquiring novel immune memories. Yet, CRISPR systems vary widely in both how and to what degree they can distinguish foreign from self-derived genetic material. We document known and hypothesized mechanisms that bias the acquisition of immune memory towards non-self targets. We demonstrate that diversity is the rule, with many widespread but no universal mechanisms for self versus non-self recognition.

Occurrence and Diversity of CRISPR Loci in Lactobacillus casei Group.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system that resists foreign genes through nuclease targeting in bacteria and archaea. In this study, we analyzed 68 strains of Lactobacillus casei group from the NCBI GenBank database, and bioinformatic tools were used to investigate the occurrence and diversity of CRISPR system. The results showed that a total of 30 CRISPR loci were identified from 27 strains. Apart from three strains which contained double loci with distinguishable distributed sites, most strains contained only one CRISPR locus. The analysis of direct repeat (DR) sequences showed that all DR could form stable RNA secondary structures. The CRISPR spacers showed diversity, and their origin and evolution were revealed through the investigation of their spacer sequences. In addition, a large number of CRISPR spacers showed perfect homologies to phage and plasmid sequences. Collectively, our results would contribute to researches of resistance in L. casei group, and also provide a new vision on the diversity and evolution of CRISPR/Cas system.

CRISPR-Cas12b enables efficient plant genome engineering.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12b is a newly emerged genome engineering system. Here, we compared Cas12b from Alicyclobacillus acidoterrestris (Aac), Alicyclobacillus acidiphilus (Aa), Bacillus thermoamylovorans (Bth) and Bacillus hisashii (Bh) for genome engineering in rice, an important crop. We found AaCas12b was more efficient than AacCas12b and BthCas12b for targeted mutagenesis, which was further demonstrated in multiplexed genome editing. We also engineered the Cas12b systems for targeted transcriptional repression and activation. Our work establishes Cas12b as the third promising CRISPR system, after Cas9 and Cas12a, for plant genome engineering.

Protein-Mutation-Induced Conformational Changes of the DNA and Nuclease Domain in CRISPR/Cas9 Systems by Molecular Dynamics Simulations.

Class 2 CRISPR (clustered regularly interspaced short palindromic repeats) systems offer a unique protocol for genome editing in eukaryotic cells. The nuclease activity of Cas9 has been harnessed to perform precise genome editing by creating double-strand breaks. However, the nuclease activity of Cas9 can be triggered when there is imperfect complementarity between the RNA guide sequence and an off-target genomic site, which is a major limitation of the CRISPR technique for practical applications. Hence, understanding the binding mechanisms in CRISPR/Cas9 for predicting ways to increase cleavage specificity is a timely research target. One way to understand and tune the binding strength is to study wild-type and mutant Cas9, in complex with a guide RNA and a target DNA. We have performed classical all-atom MD simulations over a cumulative time scale of 13.5 μs of CRISPR/Cas9 ternary complexes with the wild-type Cas9 from Streptococcus pyogenes and three of its mutants: K855A, H982A, and the combination K855A+H982A, selected from the outcome of experimental work. Our results reveal significant structural impact of the mutations, with implications for specificity. We find that the "unwound" part of the nontarget DNA strand exhibits enhanced flexibility in complexes with Cas9 mutants and tries to move away from the HNH/RuvC interface, where it is otherwise stabilized by electrostatic couplings in the wild-type complex. Our findings refine an electrostatic model by which cleavage specificity can be optimized through protein mutations.

Surpassing the detection limit and accuracy of the electrochemical DNA sensor through the application of CRISPR Cas systems

Robust developments of personalized medicine for next-generation healthcare highlight the need for sensitive and accurate point-of-care platforms for quantification of disease biomarkers. Broad presentations of clustered regularly interspaced short palindromic repeats (CRISPR) as an accurate gene editing tool also indicate that the high-specificity and programmability of CRISPR system can be utilized for the development of biosensing systems. Herein, we present a CRISPR Cas system enhanced electrochemical DNA (E-DNA) sensor with unprecedented sensitivity and accuracy. The principle of the E-DNA sensor is the target induced conformational change of the surface signaling probe (containing an electrochemical tag), leading to the variation of the electron transfer rate of the electrochemical tag. With the introduction of CRISPR cleavage activity into the E-DNA sensor, a more apparent signal change between with and without the presence of the target can be achieved. We compared the performance of Cas9 and Cas12a enhanced E-DNA sensor and optimized the chemical environment of CRISPR, achieving a femto-molar detection limit without enzymatic amplification. Moreover, we correlated the CRISPR cleavage signal with the original E-DNA signal as a strategy to indicate potential mismatches in the target sequence. Comparing with classic DNA electrochemistry based mutation detection strategy, CRISPR enhanced E-DNA sensor can determine the presence of a single mutation at an unknown concentration condition. Overall, we believe that the CRISPR enhanced E-DNA sensing strategy will be of especially high utility for point-of-care systems owing to the programmability, modularity, high-sensitivity and high-accuracy.

Formation and Stoichiometry of CRISPR-Cascade Complexes with Varying Spacer Lengths Revealed by Native Mass Spectrometry.

The adaptive immune system of bacteria and archaea against viral DNA is based on clustered, regularly interspaced, short palindromic repeats (CRISPRs) which are encoded in the host genome and translated into CRISPR RNAs (crRNAs) containing single spacer sequences complementary to foreign DNA. crRNAs assemble with CRISPR-associated (Cas) proteins forming surveillance complexes that base-pair with viral DNA and mediate its degradation. As specificity of degradation is provided by the crRNA spacer sequence, genetic engineering of the CRISPR system has emerged as a popular molecular tool, for instance, in gene silencing and programmed DNA degradation. Elongating or shortening the crRNA spacer sequence are therefore promising ventures to modify specificity toward the target DNA. However, even though the stoichiometry of wild-type complexes is well established, it is unknown how variations in crRNA spacer length affect their stoichiometry. The CRISPR-associated antiviral defense surveillance complexes of Streptococcus thermophilus (StCascade complexes) contain crRNA and five protein subunits. Using native mass spectrometry, we studied the formation and stoichiometry of StCascade complexes assembled on a set of crRNAs with different spacer lengths. We assigned all relevant complexes and gained insights into the stoichiometry of the complexes as well as their preferred assembly. We found that stable complexes, which incorporate or lose a (Cas7)2(Cse2)1-module, assemble on crRNA varied in length by 12-nucleotide units, while varying crRNA length in six-nucleotide units results in heterogeneous mixtures of complexes. Combining our results from the various variants, we generated an assembly pathway revealing general features of I-E type Cascade complex formation.

Factors Impacting Efficacy of AAV-Mediated CRISPR-Based Genome Editing for Treatment of Choroidal Neovascularization

Frequent injections of anti-vascular endothelial growth factor (anti-VEGF) agents are a clinical burden for patients with neovascular age-related macular degeneration (AMD). Genomic disruption of VEGF-A using adeno-associated viral (AAV) delivery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 has the potential to permanently suppress aberrant angiogenesis, but the factors that determine the optimal efficacy are unknown. Here, we investigate two widely used Cas9 endonucleases, SpCas9 and SaCas9, and evaluate the relative contribution of AAV-delivery efficiency and genome-editing rates in vivo to determine the mechanisms that drive successful CRISPR-based suppression of VEGF-A, using a mouse model of laser-induced choroidal neovascularization (CNV). We found that SpCas9 demonstrated higher genome-editing rates, greater VEGF reduction, and more effective CNV suppression than SaCas9, despite similar AAV transduction efficiency between a dual-vector approach for SpCas9 and single-vector system for SaCas9 to deliver the Cas9 orthologs and single guide RNAs (gRNAs). Our results suggest that successful VEGF knockdown using AAV-mediated CRISPR systems may be determined more by the efficiency of genome editing rather than viral transduction and that SpCas9 may be more effective than SaCas9 as a potential therapeutic strategy for CRISPR-based treatment of CNV in neovascular AMD.

Genomic Characterization of a Novel Gut Symbiont From the Hadal Snailfish.

Hadal trenches are characterized by not only high hydrostatic pressure but also scarcity of nutrients and high diversity of viruses. Snailfishes, as the dominant vertebrates, play an important role in hadal ecology. Although studies have suggested possible reasons for the tolerance of hadal snailfish to high hydrostatic pressure, little is known about the strategies employed by hadal snailfish to cope with low-nutrient and virus-rich conditions. In this study, the gut microbiota of hadal snailfish was investigated. A novel bacterium named “Candidatus Mycoplasma liparidae” was dominant in the guts of three snailfish individuals from both the Mariana and Yap trenches. A draft genome of “Ca. Mycoplasma liparidae” was successfully assembled with 97.8% completeness by hybrid sequencing. A set of genes encoding riboflavin biosynthesis proteins and a clustered regularly interspaced short palindromic repeats (CRISPR) system was present in the genome of “Ca. Mycoplasma liparidae,” which was unusual for Mycoplasma. The functional repertoire of the “Ca. Mycoplasma liparidae” genome is likely set to help the host in riboflavin supplementation and to provide protection against viruses via a super CRISPR system. Remarkably, genes encoding common virulence factors usually exist in Tenericutes pathogens but were lacking in the genome of “Ca. Mycoplasma liparidae.” All of these characteristics supported an essential role of “Ca. Mycoplasma liparidae” in snailfish living in the hadal zone. Our findings provide further insights into symbiotic associations in the hadal biosphere.

Conditional control of RNA-guided nucleic acid cleavage and gene editing

Prokaryotes use repetitive genomic elements termed CRISPR (clustered regularly interspaced short palindromic repeats) to destroy invading genetic molecules. Although CRISPR systems have been widely used in DNA and RNA technology, certain adverse effects do occur. For example, constitutively active CRISPR systems may lead to a certain risk of off-target effects. Here, we introduce post-synthetic masking and chemical activation of guide RNA (gRNA) to controlling CRISPR systems. An RNA structure profiling probe (2-azidomethylnicotinic acid imidazolide) is used. Moreover, we accomplish conditional control of gene editing in live cells. This proof-of-concept study demonstrates promising potential of chemical activation of gRNAs as a versatile tool for chemical biology.

CGD: Comprehensive guide designer for CRISPR-Cas systems

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems, including dead Cas9 (dCas9), Cas9, and Cas12a, have revolutionized genome engineering in mammalian somatic cells. Although computational tools that assess the target sites of CRISPR-Cas systems are inevitably important for designing efficient guide RNAs (gRNAs), they exhibit generalization issues in selecting features and do not provide optimal results in a comprehensive manner. Here, we introduce a Comprehensive Guide Designer (CGD) for four different CRISPR systems, which utilizes the machine learning algorithm, Elastic Net Logistic Regression (ENLOR), to autonomously generalize the models. CGD contains specific models trained with public datasets generated by CRISPRi, CRISPRa, CRISPR-Cas9, and CRISPR-Cas12a (designated as CGDi, CGDa, CGD9, and CGD12a, respectively) in an unbiased manner. The trained CGD models were benchmarked to other regression-based machine learning models, such as ElasticNet Linear Regression (ENLR), Random Forest and Boruta (RFB), and Extreme Gradient Boosting (Xgboost) with inbuilt feature selection. Evaluation with independent test datasets showed that CGD models outperformed the pre-existing methods in predicting the efficacy of gRNAs. All CGD source codes and datasets are available at GitHub (https://github.com/vipinmenon1989/CGD), and the CGD webserver can be accessed at http://big.hanyang.ac.kr:2195/CGD.

Phenylketonuria: Genes in phenylketonuria, diagnosis, and treatments

Introduction: Phenylketonuria (PKU) is a rare autosomal-recessive disorder inherited in accordance with the law of segregation. Detection tools for people with PKU can include Sanger Sequencing (SS) and Next Generation Sequencing (NGS). Diet therapy, Large Neutral Amino Acids (LNAA), and Specific Nutrient Combination (SNC) can help alleviate people with PKU. In the future, genetic manipulation techniques can help to eliminate PKU. Objectives: In this review, the author describes the progress in a study that focused on detection tools such as SS and NGS, the phenylalanine hydroxylase (PAH) gene and mutations in the PAH gene, use of drugs for PKU, and genetic manipulation techniques such as Adeno-associated virus (AAV) vectors and clustered regularly interspaced short palindromic repeats (CRISPR) RNA-guided FokI nuclease system (FokI-dCas9 system). AAV is abbreviation of AAV. CRISPR system is abbreviation of clustered regularly interspaced short palindromic repeats. Methods: The author searched the PubMed Database at National Center for Biotechnology Information (NCBI) for articles on PKU disorder. These articles were published between 2014 and 2019. Articles were open access and in English. Searches were also done at Google and ScienceDirect. Results: PKU derives from mutations in the PAH gene. Features of PKU may include ataxia, intellectual ability, and seizures. MassARRAY method, minisequencing method, SS and NGS can detect PKU on humans. Diet therapy, BH4, LNAA, SNC, enzyme therapy can help patients with PKU. However, these drugs cannot treat PKU permanently. In the future, genetic manipulation techniques can be used. AAV vectors and FokI-dCas9 system can be useful to eliminate PKU disorder. Conclusion: Guthrie method, MassARRAY, minisequencing, SS and NGS are tools for detecting PKU. Treatments with such as diet therapy, LNAA, SNC, and enzyme therapy are useful for PKU disorder. AAV vectors and FokI-dCas9 system are methods that can be useful for eliminating PKU in the future.

Characterization and comparison of CRISPR Loci in Streptococcus thermophilus.

Clustered regularly interspaced short palindromic repeats (CRISPR) consists of a series of regular repeat-spacer sequences. It can not only act as a natural immune system in most prokaryotes, but also be utilized as the tool of newly developed genome modification and evolutionary researches. Streptococcus thermophilus is an important model organism for the study and application of CRISPR systems. In present study, the occurrence and diversity of CRISPR-Cas systems in the genomes of S. thermophilus were investigated including 4 new sequenced strains CS5, CS9, CS18, CS20, and other 23 strains downloaded from NCBI website. 66 CRISPR/Cas systems were identified among these 27 strains and could divided into four subsystems according to the arrangement of Cas proteins, notably I-E, II-A, II-C and III-A. Overall, 26 type II-C systems, 18 type II-A systems, 13 type III-A systems, 9 type I-E systems were identified. It was mentioned that CS20 contained two type II-C systems which had not been identified in the other 26 S. thermophilus strains. Overall, 1,080 spacers were analyzed and blasted. Sequence identity searches of spacers implied that most spacers derived from partial sequences of exogenous DNA, including various bacteriophages and plasmids. Of note, a large number of novel spacers were found in this study, indicating the unique phage environment they have undergone, especially CS20 strain. In addition, the analysis of the cas1 and cas9 genes revealed the genetic relationship among CRISPR-Cas system in these strains. Furthermore, the analysis of CRISPR spacers also indicated protospacer adjacent motif (PAM) sequences. Summary of PAM sequences could lay the foundations for the application of S. thermophilus CRISPR-Cas system. Our results suggested CS5 and CS18 can be used as model strains in the research of CRISPR-Cas system, and CS20 might have greater application potential in gene editing.