RNA-Guided Genome Editing in Plants Using a CRISPR–Cas System

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a versatile tool for genetic engineering that uses cas9 to target the sequence-specific region and introduce a double-stranded break in the target area. This simple RNA-guided genome editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. This technique helps to make precise genome modification in many different tissues and organisms. Development of genetically edited crops will assist sustainable productive agriculture for better feeding of the rapidly growing population in a changing climate. The emerging areas of research for the genome editing in plants include rewiring the regulatory signaling networks, and interrogating gene functions and sgRNA library for high-throughput loss-of-function screening. This chapter deals with the strengths and weaknesses of Cas9 nuclease-mediated genome editing in plants for development of designer crops like sugarcane. With this powerful and innovative technique, the genetically engineered non-GM plants will support the sustainable agriculture and maximize yield by combating abiotic and biotic stresses.

SummaryThe Cas12j‐8 nuclease, derived from the type V CRISPR system, is approximately half the size of Cas9 and recognizes a 5′‐TTN‐3′ protospacer adjacent motif sequence, thus potentially having broad application in genome editing for crop improvement. However, its editing efficiency remains low in plants. In this study, we rationally engineered both the crRNA and the Cas12j‐8 nuclease. The engineered crRNA and Cas12j‐8 markedly improved genome editing efficiency in plants. When combined, they exhibited robust editing activity in soybean and rice, enabling the editing of target sites that were previously uneditable. Notably, for certain target sequences, the editing activity was comparable to that of SpCas9 when targeting identical sequences, and it outperformed the Cas12j‐2 variant, nCas12j‐2, across all tested targets. Additionally, we developed cytosine base editors based on the engineered crRNA and Cas12j‐8, demonstrating an average increase of 5.36‐ to 6.85‐fold in base‐editing efficiency (C to T) compared with the unengineered system in plants, with no insertions or deletions (indels) observed. Collectively, these findings indicate that the engineered hypercompact CRISPR/Cas12j‐8 system serves as an efficient tool for genome editing mediated by both nuclease cleavage and base editing in plants.

Progresses of CRISPR/Cas9 genome editing in forage crops

Biotic and abiotic stress affects the productivity and quality of the plant; it is necessarily important to develop improved crops that can withstand the environmental conditions. Conventional methods of breeding are very tedious process that require some alternative. The molecular approach of genetic modification emerged out as a precise method for plant breeding in the past few years. Genome is altered by removing, adding or substituting DNA bases in the host genome. Different methods of genome editing have been developed, including transcription activator like effector nucleases (TALENs), zinc-finger nucleases (ZFNs) and Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas. These nucleases have enabled researchers to manipulate gene of interest in crop plants. Among these CRISPR-Cas has been developed recently and found to be a more efficient tool for genome editing because of its simple design and low cost. In recent years, CRISPR-Cas9 has been utilized for gene knockouts and knock-ins, multiplex gene targeting, and gene regulation in plants. In this review, we provide the use of CRISPR–Cas9 technology in genome editing and its fruitful applications in plants specifically in terms of improvement in quality and resistance to biotic and abiotic stress. Designing of an efficient CRISPR–Cas system using bioinformatic tools have also been discussed.

Chapter 7 - CRISPR technology for genome editing

The autobiographical genre is a problem when it is considered inside the theatral genre. Because the dramatical author express himself through the mouth of fictional characters, the words of the characters could not be considered as the expression of the author himself. However, it seems that through the dialogues that can be heard, the situations, the characters and the places that are staged, some plays, especially since the 18th century, which represents a major step in self-expression, contains the signs of an autobiographical expression. Although the autobiographical genre as defined by P. Lejeune or G. Gusdorf does not exists in the theatre genre, we will study here how it nevertheless expresses itself, that is to say almost always in a hidden, implicit, ambiguous, diverted, but probable way. We will think of a method allowing the reader, but also the member of the audience, to identify and testify the presence of this autobiographical expression by emphazising some textual and scenic aspects. We will also work on connecting the autobiographical expression with the relationship that live the authors of the new work contained in our corpus with the audience. This relationship being fully opend up in emotion. Some feelings, coming with physical reactions felt by the reader/audience, are created by the recognition of the author's voice in a drama that mixes autobiographical and fiction. We will take an interest in the autobiographical expression of the dramatical author through the perspective of his particular relationship with the audience, relationship that contrasts with Brecht's principles, which have ruled the theatrical creation in the 20th century.

BackgroundThe RNA-guided Cas9 system represents a flexible approach for genome editing in plants. This method can create specific mutations that knock-out or alter target gene function. It provides a valuable tool for plant research and offers opportunities for crop improvement.ResultsWe investigate the use and target specificity requirements of RNA-guided Cas9 genome editing in barley (Hordeum vulgare) and Brassica oleracea by targeting multicopy genes. In barley, we target two copies of HvPM19 and observe Cas9-induced mutations in the first generation of 23 % and 10 % of the lines, respectively. In B. oleracea, targeting of BolC.GA4.a leads to Cas9-induced mutations in 10 % of first generation plants screened. In addition, a phenotypic screen identifies T0 plants with the expected dwarf phenotype associated with knock-out of the target gene. In both barley and B. oleracea stable Cas9-induced mutations are transmitted to T2 plants independently of the T-DNA construct. We observe off-target activity in both species, despite the presence of at least one mismatch between the single guide RNA and the non-target gene sequences. In barley, a transgene-free plant has concurrent mutations in the target and non-target copies of HvPM19.ConclusionsWe demonstrate the use of RNA-guided Cas9 to generate mutations in target genes of both barley and B. oleracea and show stable transmission of these mutations thus establishing the potential for rapid characterisation of gene function in these species. In addition, the off-target effects reported offer both potential difficulties and specific opportunities to target members of multigene families in crops.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0826-7) contains supplementary material, which is available to authorized users.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has enabled efficient, markerless genome editing in a wide range of organisms. However, there is an off-target effect and a limit to the area of precise editing. Bases that can be precisely edited are limited to within the 20-base pair gRNA-targeting site and protospacer adjacent motif (PAM) sequence. We have developed a CRISPR nickase system that can perform a precise genome-wide base editing in Saccharomyces cerevisiae using a single Cas9 nickase. This system can precisely edit a broader genomic region by the avoidance of double-strand break (DSB) and subsequent non-homologous end joining (NHEJ). Furthermore, unintended mutations were not found at off-target sites in this system. In combination with yeast gap repair cloning, precise genome editing of yeast cells can be performed in 5days. Here, we describe the methods for precise and convenient genome editing using this novel CRISPR nickase system.

The development of novel genome editing tools has unlocked new opportunities that were not previously possible in basic and biomedical research. During the last two decades, several new genome editing methods have been developed that can be customized to modify specific regions of the genome. However, in the past couple of years, many newer and more exciting genome editing techniques have been developed that are more efficient, precise, and easier to use. These genome editing tools have helped to improve our understanding of genetic disorders by modeling them in cells and animal models, in addition to correcting the disease-causing mutations. Among the genome editing tools, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has proven to be the most popular one due to its versatility and has been successfully used in a wide variety of laboratory animal models and plants. In this review, we summarize the customizable nucleases currently used for genome editing and their uses beyond the modification of genome. We also discuss the potential future applications of gene editing tools for both basic research and clinical purposes.

New prospects on the horizon: Genome editing to engineer plants for desirable traits

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity have become a household name among biologists and even the general public thanks to the unprecedented success of the new generation of genome editing tools utilizing Cas proteins. However, the fundamental biological features of CRISPR-Cas are of no lesser interest and have major impacts on our understanding of the evolution of antivirus defense, host-parasite coevolution, self versus non-self discrimination and mechanisms of adaptation. CRISPR-Cas systems present the best known case in point for Lamarckian evolution, i.e. generation of heritable, adaptive genomic changes in response to encounters with external factors, in this case, foreign nucleic acids. CRISPR-Cas systems employ multiple mechanisms of self versus non-self discrimination but, as is the case with immune systems in general, are nevertheless costly because autoimmunity cannot be eliminated completely. In addition to the autoimmunity, the fitness cost of CRISPR-Cas systems appears to be determined by their inhibitory effect on horizontal gene transfer, curtailing evolutionary innovation. Hence the dynamic evolution of CRISPR-Cas loci that are frequently lost and (re)acquired by archaea and bacteria. Another fundamental biological feature of CRISPR-Cas is its intimate connection with programmed cell death and dormancy induction in microbes. In this and, possibly, other immune systems, active immune response appears to be coupled to a different form of defense, namely, “altruistic” shutdown of cellular functions resulting in protection of neighboring cells. Finally, analysis of the evolutionary connections of Cas proteins reveals multiple contributions of mobile genetic elements (MGE) to the origin of various components of CRISPR-Cas systems, furthermore, different biological systems that function by genome manipulation appear to have evolved convergently from unrelated MGE. The shared features of adaptive defense systems and MGE, namely the ability to recognize and cleave unique sites in genomes, make them ideal candidates for genome editing and engineering tools.

The laboratory rat has been widely used as an animal model in biomedical science for more than 150 years. Applying zinc-finger nucleases or transcription activator-like effector nucleases to rat embryos via microinjection is an efficient genome editing tool for generating targeted knockout rats. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonucleases have been used as an effective tool for precise and multiplex genome editing in mice and rats. In this review, the advantages and disadvantages of these site-specific nuclease technologies for genetic analysis and manipulation in rats are discussed.

Recent Progress in CRISPR/Cas9 Technology

Finding and explaining the functions of genes in plants have promising applications in crop improvement and bioprospecting and hence, it is important to compare various techniques available for gene function identification in plants. Today, the most popular technology among researchers to identify the functions of genes is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-based genome editing method. But by no means can we say that CRISPR/Cas9 is the go-to method for all purposes. It comes with its own baggage. Researchers will agree and have lived through at least seven more technologies deployed to find the functions of genes, which come under three umbrellas: 1. genetic engineering, 2. transient expression, and 3. chemical/physical mutagenesis. Each of the methods evolved when the previous one ran into an insurmountable problem. In this review, we compare the eight technologies against one another on 14 parameters. This review lays bare the pros and cons, and similarities and dissimilarities of various methods. Every method comes with its advantages and disadvantages. For example, the CRISPR/Cas9-based genome editing is an excellent method for modifying gene sequences, creating allelic versions of genes, thereby aiding the understanding of gene function. But it comes with the baggage of unwanted or off-target mutations. Then, we have methods based on random or targeted knockout of the gene, knockdown, and overexpression of the gene. Targeted disruption of genes is required for complete knockout of gene function, which may not be accomplished by editing. We have also discussed the strategies to overcome the shortcomings of the targeted gene-knockout and the CRISPR/Cas9-based methods. This review serves as a comprehensive guide towards the understanding and comparison of various technologies available for gene function identification in plants and hence, it will find application for crop improvement and bioprospecting related research.

The major goal of animal breeding is the genetic enhancement of economic traits. The CRISPR/Cas system, which includes nuclease-mediated and base editor mediated genome editing tools, provides an unprecedented approach to modify the mammalian genome. Thus, farm animal genetic engineering and genetic manipulation have been fundamentally revolutionized. Agricultural animals with traits of interest can be obtained in just one generation (and without long time selection). Here, we reviewed the advancements of the CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated proteins) genome editing tools and their applications in animal breeding, especially in improving disease resistance, production performance, and animal welfare. Additionally, we covered the regulations on genome-edited animals (GEAs) and ways to accelerate their use. Recommendations for how to produce GEAs were also discussed. Despite the current challenges, we believe that genome editing breeding and GEAs will be available in the near future.