Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster ...regularly interspaced short palindromic repeats （CRISPR）-associated nuclease （Cas） is emerging as an efficient tool for genome editing in microbial and animal systems. Here, we report the genome editing and targeted gene mutation in plants via the CRISPR-Cas9 sys- tem. Three guide RNAs （gRNAs） with a 20-22-nt seed region were designed to pair with distinct rice genomic sites which are followed by the protospacer-adjacent motif （PAM）. The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired sites and introduce mutation （insertion or deletion） by error-prone non-homologous end joining DNA repairing. By analyzing the RNA-guided genome-editing events, the mutation efficiency at these target sites was estimated to be 3-8%. In addition, the off-target effect of an engineered gRNA-Cas9 was found on an imper- fectly paired genomic site, but it had lower genome-editing efficiency than the perfectly matched site. Further analysis suggests that mismatch position between gRNA seed and target DNA is an important determinant of the gRNA-Cas9 tar- geting specificity, and specific gRNAs could be designed to target more than 90% of rice genes. Our results demonstrate that the CRISPR-Cas system can be exploited as a powerful tool for gene targeting and precise genome editing in plants.
International treaties, conventions, and organizations to protect refugees were established in the aftermath of World War II to protect people escaping targeted persecution by their own governments. ...However, the nature of cross-border displacement has transformed dramatically since then. Such threats as environmental change, food insecurity, and generalized violence force massive numbers of people to flee states that are unable or unwilling to ensure their basic rights, as do conditions in failed and fragile states that make possible human rights deprivations. Because these reasons do not meet the legal understanding of persecution, the victims of these circumstances are not usually recognized as "refugees," preventing current institutions from ensuring their protection. In this book, Alexander Betts develops the concept of "survival migration" to highlight the crisis in which these people find themselves.
Examining flight from three of the most fragile states in Africa-Zimbabwe, the Democratic Republic of Congo, and Somalia-Betts explains variation in institutional responses across the neighboring host states. There is massive inconsistency. Some survival migrants are offered asylum as refugees; others are rounded up, detained, and deported, often in brutal conditions. The inadequacies of the current refugee regime are a disaster for human rights and gravely threaten international security. InSurvival Migration, Betts outlines these failings, illustrates the enormous human suffering that results, and argues strongly for an expansion of protected categories.
Beyond their widespread application as genome‐editing and regulatory tools, clustered regularly interspaced short palindromic repeats (CRISPR)‐CRISPR‐associated (Cas) systems also play a critical ...role in nucleic acid detection due to their high sensitivity and specificity. Recently developed Cas family effectors have opened the door to the development of new strategies for detecting different types of nucleic acids for a variety of purposes. Precise and efficient nucleic acid detection using CRISPR‐Cas systems has the potential to advance both basic and applied biological research. In this review, we summarize the CRISPR‐Cas systems used for the recognition and detection of specific nucleic acids for different purposes, including the detection of genomic DNA, nongenomic DNA, RNA, and pathogenic microbe genomes. Current challenges and further applications of CRISPR‐based detection methods will be discussed according to the most recent developments.
This review highlights strategies to detect nucleic acids with high sensitivity and selectivity based on different CRISPR‐Cas systems, emphasizing on the detection of genomic DNA, nongenomic DNA, RNA and pathogenic microbe genomes. The outstanding challenges and future scientific directions ripe for the CRISPR‐based nucleic acid detection techniques were finally discussed.
The CRISPR/Cas technology is enabling targeted genome editing in multiple organisms with unprecedented accuracy and specificity by using RNA-guided nucleases. A critical point when planning a ...CRISPR/Cas experiment is the design of the guide RNA (gRNA), which directs the nuclease and associated machinery to the desired genomic location. This gRNA has to fulfil the requirements of the nuclease and lack homology with other genome sites that could lead to off-target effects. Here we introduce the Breaking-Cas system for the design of gRNAs for CRISPR/Cas experiments, including those based in the Cas9 nuclease as well as others recently introduced. The server has unique features not available in other tools, including the possibility of using all eukaryotic genomes available in ENSEMBL (currently around 700), placing variable PAM sequences at 5' or 3' and setting the guide RNA length and the scores per nucleotides. It can be freely accessed at: http://bioinfogp.cnb.csic.es/tools/breakingcas, and the code is available upon request.
Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and ...even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 3' GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome.
Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. ...Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small non-coding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.
Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle ...to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications, including rewiring cell fates or engineering metabolic pathways.
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•CRISPR scaffold RNAs (scRNAs) encode both target locus and regulatory function•scRNAs function efficiently in mammalian and yeast cells•scRNAs enable transcription programs with simultaneous activation and repression•Combinatorial control of multiple genes enables flexible pathway manipulation
Modular CRISPR RNA scaffolds engineered to encode both guides to a target locus and recruitment of transcriptional regulators allow simultaneous gene activation and repression of multiple different genes in eukaryotic cells, greatly expanding the synthetic biology toolkit.
Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely ...employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.
The CRISPR/Cas9-sgRNA system has been developed to mediate genome editing and become a powerful tool for biological research. Employing the CRISPR/Cas9-sgRNA system for genome editing and ...manipulation has accelerated research and expanded researchers' ability to generate genetic models. However, the method evaluating the efficiency of sgRNAs is lacking in plants. Based on the nucleotide compositions and secondary structures of sgRNAs which have been experimentally validated in plants, we instituted criteria to design efficient sgRNAs. To facilitate the assembly of multiple sgRNA cassettes, we also developed a new strategy to rapidly construct CRISPR/Cas9-sgRNA system for multiplex editing in plants. In theory, up to ten single guide RNA (sgRNA) cassettes can be simultaneously assembled into the final binary vectors. As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed. Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency. This work provides a convenient approach to select efficient sgRNAs for target editing.
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