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Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3'-terminal segment of guide RNA.
CRISPR-Cas9 is widely applied for genome engineering in various organisms. The assembly of single guide RNA (sgRNA) with the Cas9 protein may limit the Cas9/sgRNA effector complex function. We developed a FRET-based assay for detection of CRISPR-Cas9 complex binding to its targets and used this assay to investigate the kinetics of Cas9 assembly with a set of structurally distinct sgRNAs. We find that Cas9 and isolated sgRNAs form the effector complex efficiently and rapidly. Yet, the assembly process is sensitive to the presence of moderate concentrations of non-specific RNA competitors, which considerably delay the Cas9/sgRNA complex formation, while not significantly affecting already formed complexes. This observation suggests that the rate of sgRNA loading into Cas9 in cells can be determined by competition between sgRNA and intracellular RNA molecules for the binding to Cas9. Non-specific RNAs exerted particularly large inhibitory effects on formation of Cas9 complexes with sgRNAs bearing shortened 3'-terminal segments. This result implies that the 3'-terminal segment confers sgRNA the ability to withstand competition from non-specific RNA and at least in part may explain the fact that use of sgRNAs truncated for the 3'-terminal stem loops leads to reduced activity during genomic editing.
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Utilizing Target Sequences with Multiple Flanking Protospacer Adjacent Motif (PAM) Sites Reduces Off-Target Effects of the Cas9 Enzyme in Pineapple.
BACKGROUND/OBJECTIVES: CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)-associated protein 9 is now widely used in agriculture and medicine. Off-target effects can lead to unexpected results that may be harmful, and these effects are a common concern in both research and therapeutic applications. METHODS: In this study, using pineapple as the gene-editing material, eighteen target sequences with varying numbers of PAM (Protospacer-Adjacent Motif) sites were used to construct gRNA vectors. Fifty mutant lines were generated for each target sequence, and the off-target rates were counted. RESULTS: Selecting sequences with multiple flanking PAM sites as editing targets resulted in a lower off-target rate compared to those with a single PAM site. Target sequences with two 5'-NGG ("N" represents any nucleobase, followed by two guanine "G") PAM sites at the 3' end exhibited greater specificity and a higher probability of binding with the Cas9 protein than those only with one 5'-NGG PAM site at the 3' end. Conversely, although the target sequence with a 5'-NAG PAM site (where "N" is any nucleobase, followed by adenine "A" and guanine "G") adjacent and upstream of an NGG PAM site had a lower off-target rate compared to sequences with only an NGG PAM site, their off-target rates were still higher than those of sequences with two adjacent 5'-NAG PAM sites. Among the target sequences of pineapple mutant lines (AcACS1, AcOT5, AcCSPE6, AcPKG11A), more deletions than insertions were found. CONCLUSIONS: We found that target sequences with multiple flanking PAM sites are more likely to bind with the Cas9 protein and induce mutations. Selecting sequences with multiple flanking PAM sites as editing targets can reduce the off-target effects of the Cas9 enzyme in pineapple. These findings provide a foundation for improving off-target prediction and engineering CRISPR-Cas9 complexes for gene editing.
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The landscape of CRISPR/Cas9 for inborn errors of metabolism.
Since its discovery as a genome editing tool, the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) system has opened new horizons in the diagnosis, research, and treatment of genetic diseases. CRISPR/Cas9 can rewrite the genome at any region with outstanding precision to modify it and further instructions for gene expression. Inborn Errors of Metabolism (IEM) are a group of more than 1500 diseases produced by mutations in genes encoding for proteins that participate in metabolic pathways. IEM involves small molecules, energetic deficits, or complex molecules diseases, which may be susceptible to be treated with this novel tool. In recent years, potential therapeutic approaches have been attempted, and new models have been developed using CRISPR/Cas9. In this review, we summarize the most relevant findings in the scientific literature about the implementation of CRISPR/Cas9 in IEM and discuss the future use of CRISPR/Cas9 to modify epigenetic markers, which seem to play a critical role in the context of IEM. The current delivery strategies of CRISPR/Cas9 are also discussed.
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Improving CRISPR-Cas9 On-Target Specificity.
The CRISPR-Cas9 has revolutionized the field of molecular biology, medical genetics and medicine. The technology is robust, facile and simple to achieve genome targeting in cells and organisms. However, to propagate these nucleases for therapeutic application, the on-target specificity is of paramount importance. Although the binding and cleavage of off-target sites by Cas9 is issue of concern, however the specificity of CRISPR technology is greatly improved in current research employing the use of engineer nucleases, improved gRNA selection, novel Cas9 orhtologs and the advancement in methods to detect and screen off-target sites and its effects. Here we summarize the advances in this state-of-the-art technology that will equip the genome editing tools to be applied in clinical research. The researcher should optimize these methods with emphasize to achieve perfection in the specificity.
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The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny.
The CRISPR/Cas nuclease is becoming a major tool for targeted mutagenesis in eukaryotes by inducing double-strand breaks (DSBs) at pre-selected genomic sites that are repaired by non-homologous end joining (NHEJ) in an error-prone way. In plants, it could be demonstrated that the Cas9 nuclease is able to induce heritable mutations in Arabidopsis thaliana and rice. Gene targeting (GT) by homologous recombination (HR) can also be induced by DSBs. Using a natural nuclease and marker genes, we previously developed an in planta GT strategy in which both a targeting vector and targeting locus are activated simultaneously via DSB induction during plant development. Here, we demonstrate that this strategy can be used for natural genes by CRISPR/Cas-mediated DSB induction. We were able to integrate a resistance cassette into the ADH1 locus of A. thaliana via HR. Heritable events were identified using a PCR-based genotyping approach, characterised by Southern blotting and confirmed on the sequence level. A major concern is the specificity of the CRISPR/Cas nucleases. Off-target effects might be avoided using two adjacent sgRNA target sequences to guide the Cas9 nickase to each of the two DNA strands, resulting in the formation of a DSB. By amplicon deep sequencing, we demonstrate that this Cas9 paired nickase strategy has a mutagenic potential comparable with that of the nuclease, while the resulting mutations are mostly deletions. We also demonstrate the stable inheritance of such mutations in A. thaliana.
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CRISPR/Cas9-Mediated Genetic Engineering to Generate a Disease Model Prairie Vole, Based on Species-Optimized Assisted Reproductive Technology.
Social and prosocial behaviors, including communication, social bonding, and affiliation, parental behaviors, and empathy are key features of a highly social mammalian species. However, the neuronal mechanism in the brain underlying these behaviors remains unclear because of limited information on the social and prosocial behavioral levels in rodent models generally used in behavioral neuroscience studies.The rodent species, prairie vole (Microtus ochrogaster), is one of the nontraditional animal models with several advantages in experimental science over other rodent models, such as mice or rats. Additionally, it demonstrates characteristics advantageous in the study of social and prosocial behaviors, such as monogamous pair bonding behavior, biparental care, and consoling behavior toward partners stressed by aversive foot shock stimulus. Recent studies of prairie voles have highlighted the importance of oxytocin (OXT) and oxytocin receptor (OXTR)-mediated mechanisms in the regulation of these behaviors.Recently, we established assisted reproductive technologies for prairie voles, and successfully and efficiently generated an OXTR gene knockout (KO) prairie vole using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9 ), a powerful genome editing tool with artificially developed single-strand guide RNAs (sgRNA) and Cas9 endonucleases.Herein, we describe the method for CRISPR /Cas9-mediated generation of OXTR KO prairie vole. This OXTR KO prairie vole can be a valuable tool to understand their unique social and prosocial behaviors and elucidate how the oxytocin system influences or modulates these behaviors in the brain.
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Recent developments and applications of genetic transformation and genome editing technologies in wheat.
Wheat (Triticum aestivum) is a staple crop across the world and plays a remarkable role in food supplying security. Over the past few decades, basic and applied research on wheat has lagged behind other cereal crops due to the complex and polyploid genome and difficulties in genetic transformation. A breakthrough called as PureWheat was made in the genetic transformation of wheat in 2014 in Asia, leading to a noticeable progress of wheat genome editing. Due to this great achievement, it is predicated that wheat biotechnology revolution is arriving. Genome editing technologies using zinc finger nucleases, transcription activator-like effector nuclease, and clustered regularly interspaced short palindromic repeats-associated endonucleases (CRISR/Cas) are becoming powerful tools for crop modification which can help biologists and biotechnologists better understand the processes of mutagenesis and genomic alteration. Among the three genome editing systems, CRISR/Cas has high specificity and activity, and therefore it is widely used in genetic engineering. Generally, the genome editing technologies depend on an efficient genetic transformation system. In this paper, we summarize recent progresses and applications on genetic transformation and genome editing in wheat. We also examine the future aspects of genetic transformation and genome editing. We believe that the technologies for wheat efficient genetic engineering and functional studies will become routine with the emergence of high-quality genomic sequences.
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Production of genome-edited pluripotent stem cells and mice by CRISPR/Cas.
Clustered regularly at interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases, so-called CRISPR/Cas, was recently developed as an epoch-making genome engineering technology. This system only requires Cas9 nuclease and single-guide RNA complementary to a target locus. CRISPR/Cas enables the generation of knockout cells and animals in a single step. This system can also be used to generate multiple mutations and knockin in a single step, which is not possible using other methods. In this review, we provide an overview of genome editing by CRISPR/Cas in pluripotent stem cells and mice.
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Application of the CRISPR/Cas system for genome editing in microalgae.
Microalgae are arguably the most abundant single-celled eukaryotes and are widely distributed in oceans and freshwater lakes. Moreover, microalgae are widely used in biotechnology to produce bioenergy and high-value products such as polyunsaturated fatty acids (PUFAs), bioactive peptides, proteins, antioxidants and so on. In general, genetic editing techniques were adapted to increase the production of microalgal metabolites. The main genome editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system. Due to its high genome editing efficiency, the CRISPR/Cas system is emerging as the most important genome editing method. In this review, we summarized the available literature on the application of CRISPR/Cas in microalgal genetic engineering, including transformation methods, strategies for the expression of Cas9 and sgRNA, the CRISPR/Cas9-mediated gene knock-in/knock-out strategies, and CRISPR interference expression modification strategies.
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B-cell editing: An emerging alternative of precision oncotherapy.
Lately, the urgency of precision medicine in cancer care through immunotherapy has reformed the arena of oncology. Although immunomodulatory therapeutics in cancer have been preliminarily concentrated on T-cells, emerging evidences have suggested that intra-tumoral B-cells and plasma cells have significant contributions in cancer prognosis primarily through the production of antibodies. B-cell oriented cancer vaccines have been used in early clinical trials of breast and other cancers after multiple preclinical studies. Passive immunotherapy via administration of monoclonal antibodies (mAbs) and emergence of anti-idiotypic antibodies have led to considerable advancement in oncotherapy. Endogenous production of mAbs would be of significant benefit in recurrent or residual malignancies and permanent infusion would help in the overcoming of issues related to pharmacodynamic variations observed in case of intravenous inoculations of bi or tri specific mAbs. This has directed towards the development of genome reprogrammed B-cells with the capability of yielding therapeutic mAbs independently. Genetic alteration through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) nucleases have enabled the introduction of transgenes into B-cell genome thereby stimulating the plasma cells to produce exogenous remedial antibodies. It also facilitates ex vivo B-cell editing to elevate specificities of antigen receptors and generate target specific antibody responses which cannot normally be evoked in patient's immune system. Hence, genome-altered B-cells possess the potential of engineered therapeutics against certain malignancies. Co-operation of B-cells in T-cell based vaccines are ultimate need for vaccine success. In this chapter, the mechanisms, challenges and potential advantages of B-cell editing in cancer immune therapy shall be explored. The prospects of B-cell editing in onco-therapy will be clearly elucidated with all its strength and weaknesses.
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