Targeted genome modification of crop plants using a CRISPR-Cas system

Crop improvement aims at substantial enhancements in the quality, yield and stress resistance of crops to meet the increasing food demand of growing world population. Targeted genome modification of crop plants is one of the ways to achieve this. This technology supersedes conventional methods limited by the inefficiencies of random mutation, accuracy and stability. It employs site-directed nucleases to create breaks at specific points in the target genome for desired alteration with high-precision. There are four nucleases namely, LAGLIDADG homing endonucleases (LHEs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR) nucleases out of which three, ZFNs, TALENs and CRISPR have been highly studied and evaluated in various crop systems for economic trait. Potency of engineered nucleases lies in their efficacy to bring desired modification in diploid as well as in polyploid plant genomes. Modifications using genome editing are similar to natural or conventional method like induced mutations and are foreseen to waive regulatory actions as applicable to genetically modified organisms. This review seeks to emphasize on the employment of engineered nucleases in various crops plants till date.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) explain genetic illness and how people can treat it using. CRISPR, a gene editing technology, has altered what is now possible in animal modification and the development of human treatments. Technological advancements enable new enhanced plants, breakthrough concepts for human medicine, and appealing yet feasible techniques for reducing vector-borne illnesses. CRISPR is used as a diagnostic method for several critical diseases like cancer. CRISPR can detect and identify the DNA and RNA to identify the cause of the pathogen, like viruses or bacteria with high sensitivity. In this research, the researcher will explain how CRISPR will change the future, specifically for medical purposes. The researcher will do the Systematic Literature Review (SLR) to describe CRISPR. The goal considering CRISPR in the future is routinely used to edit the genetics of plant, bacterial, and even animal models for good purposes. It also nourishes and protects the human body from diseases by examining target genes in genome modification, investigating, and treating genetic disorders, infectious diseases, and immunological diseases. In CRISPR applications for hereditary illnesses, the CRISPR/Cas technology has been used for gene therapy to protect humans against sickness. Although the limitation of the technology is that still in the initial stages, CRISPR could be one of the groundbreaking methods in the future.

Genome engineering by site-specific nucleases enables reverse genetics and targeted editing of genomes in an efficacious manner. Contemporary revolutionized progress in targeted-genome engineering technologies based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-related RNA-guided endonucleases facilitate coherent interrogation of crop genome function. Evolved as an innate component of the adaptive immune response in bacterial and archaeal systems, CRISPR/Cas system is now identified as a versatile molecular tool that ensures specific and targeted genome modification in plants. Applications of this genome redaction tool-kit include somatic genome editing, rectification of genetic disorders or gene therapy, treatment of infectious diseases, generation of animal models, and crop improvement. We review the utilization of these synthetic nucleases as precision, targeted-genome editing platforms with the inherent potential to accentuate basic science “strengths and shortcomings” of gene function, complement plant breeding techniques for crop improvement, and charter a knowledge base for effective use of editing technology for ever-increasing agricultural demands. Furthermore, the emerging importance of Cpf1, Cas9 nickase, C2c2, as well as other innovative candidates that may prove more effective in driving novel applications in crops are also discussed. The mined data has been prepared as a library and opened for public use at www.lipre.org.

Genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by sequence-specific nucleases (SSNs) to initiate DNA repair reactions that are based on either non-homologous end joining (NHEJ) or homology-directed repair (HDR). While complete knock-outs and loss-of-function mutations generated by NHEJ are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by random point mutations or indels at specific loci in either the genes’ encoding or promoter regions. Therefore, genome modification through SSNs-mediated HDR for gene targeting (GT) that enables either gene replacement or knock-in will provide an unprecedented ability to facilitate plant breeding by allowing introduction of precise point mutations and new gene functions, or integration of foreign genes at specific and desired “safe” harbor in a predefined manner. The emergence of three programmable SSNs, such as zinc finger nucleases, transcriptional activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems has revolutionized genome modification in plants in a more controlled manner. However, while targeted mutagenesis is becoming routine in plants, the potential of GT technology has not been well realized for traits improvement in crops, mainly due to the fact that NHEJ predominates DNA repair process in somatic cells and competes with the HDR pathway, and thus HDR-mediated GT is a relative rare event in plants. Here, we review recent research findings mainly focusing on development and applications of precise GT in plants using three SSNs systems described above, and the potential mechanisms underlying HDR events in plant cells. We then address the challenges and propose future perspectives in order to facilitate the implementation of precise genome modification through SSNs-mediated GT for crop improvement in a global context.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a prokaryotic adaptive immune system that has the ability to identify specific locations on the bacteriophage (phage) genome to create breaks in it, and internalize the phage genome fragments in its own genome as CRISPR arrays for memory-dependent resistance. Although CRISPR has been used in the dairy industry for a long time, it recently gained importance in the field of genome editing because of its ability to precisely target locations in a genome. This system has further been modified to locate and target any region of a genome of choice due to modifications in the components of the system. By changing the nucleotide sequence of the 20-nucleotide target sequence in the guide RNA, targeting any location is possible. It has found an application in the modification of plant genomes with its ability to generate mutations and insertions, thus helping to create new varieties of plants. With the ability to introduce specific sequences into the plant genome after cleavage by the CRISPR system and subsequent DNA repair through homology-directed repair (HDR), CRISPR ensures that genome editing can be successfully applied in plants, thus generating stronger and more improved traits. Also, the use of the CRISPR editing system can generate plants that are transgene-free and have mutations that are stably inherited, thus helping to circumvent current GMO regulations.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated protein) system, a prokaryotic RNA-based adaptive immune system against viral infection, is emerging as a powerful genome editing tool in broad research areas. To further improve and expand its functionality, various CRISPR delivery strategies have been tested and optimized, and key CRISPR system components such as Cas protein have been engineered with different purposes. Benefiting from more in-depth understanding and further development of CRISPR, versatile CRISPR-based platforms for genome editing have been rapidly developed to advance investigations in biology and biomedicine. In biological research area, CRISPR has been widely adopted in both fundamental and applied research fields, such as genomic and epigenomic modification, genome-wide screening, cell and animal research, agriculture transforming, livestock breeding, food manufacture, industrial biotechnology, and gene drives in disease agents control. In biomedical research area, CRISPR has also shown its extensive applicability in the establishment of animal models for genetic disorders, generation of tissue donors, implementation of antimicrobial and antiviral studies, identification and assessment of new drugs, and even treatment for clinical diseases. However, there are still several problems to consider, and the biggest concerns are the off-target effects and ethical issues of this technology. In this prospect article, after highlighting recent development of CRISPR systems, we outline different applications and current limitations of CRISPR in biological and biomedical investigation. Finally, we provide a perspective on future development and potential risks of this multifunctional technology. J. Cell. Biochem. 119: 52-61, 2018. © 2017 Wiley Periodicals, Inc.

The Streptococcus-derived CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) system has emerged as a very powerful tool for targeted gene modifications in many living organisms including plants. Since the first application of this system for plant gene modification in 2013, this RNA-guided DNA endonuclease system has been extensively engineered to meet the requirements of functional genomics and crop trait improvement in a number of plant species. Given its short history, the emphasis of many studies has been the optimization of the technology to improve its reliability and efficiency to generate heritable gene modifications in plants. Here we review and analyze the features of customized CRISPR/Cas9 systems developed for plant genetic studies and crop breeding. We focus on two essential aspects: the heritability of gene modifications induced by CRISPR/Cas9 and the factors affecting its efficiency, and we provide strategies for future design of systems with improved activity and heritability in plants.

Genome editing is an approach in which a specific target DNA sequence of the genome is altered by adding, removing, or replacing DNA bases. Artificially engineered hybrid enzymes, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), and the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) system are being used for genome editing in various organisms including plants. The CRISPR-Cas system has been developed most recently and seems to be more efficient and less time-consuming compared with ZFNs or TALENs. This system employs an RNA-guided nuclease, Cas9, to induce double-strand breaks. The Cas9-mediated breaks are repaired by cellular DNA repair mechanisms and mediate gene/genome modifications. Here, we provide a detailed overview of the CRISPR-Cas system and its adoption in different organisms, especially plants, for various applications. Important considerations and future opportunities for deployment of the CRISPR-Cas system in plants for numerous applications are also discussed. Recent investigations have revealed the implications of the CRISPR-Cas system as a promising tool for targeted genetic modifications in plants. This technology is likely to be more commonly adopted in plant functional genomics studies and crop improvement in the near future.

Concerns regarding ‘off-target’ activity of genome editing endonucleases

Over the last few years, the use of clustered regularly interspaced short palindromic repeats (CRISPR) for genetic manipulation has transformed life science. CRISPR was first found in bacteria and archaea as an adaptable immune system, and later modified to create specific DNA breaks in living cells and creatures. Various DNA alterations can occur throughout the cellular DNA repair process. Since the first demonstration of CRISPR in plant genome editing in 2013, there has been much progress in fundamental crop research and plant improvement. Plants can use the CRISPR toolset to do programmable genome editing, epigenome editing, and transcriptome regulation. However, the difficulties of plant genome editing must be properly understood and answers sought. With an emphasis on achievements and prospective utility in plant biology, this review aims to provide an instructive assessment of the current advancements and discoveries in CRISPR technology. CRISPR will, in the end, not only make fundamental research easier, but it will also speed up plant breeding and germplasm development. In the light of global climate change, as well as present agricultural, environmental, and ecological concerns, the use of CRISPR to improve germplasm is extremely significant.

Targeted modification of plant genome is key for elucidating and manipulating gene functions in basic and applied plant research. The CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein (Cas) technology is emerging as a powerful genome editing tool in diverse organisms. This technology utilizes an easily reprogrammable guide RNA (gRNA) to guide Streptococcus pyogenes Cas9 endonuclease to generate a DNA double-strand break (DSB) within an intended genomic sequence and subsequently stimulate chromosomal mutagenesis or homologous recombination near the DSB site through cellular DNA repair machineries. In this chapter, we describe the detailed procedure to design, construct, and evaluate dual gRNAs for plant codon-optimized Cas9 (pcoCas9)-mediated genome editing using Arabidopsis thaliana and Nicotiana benthamiana protoplasts as model cellular systems. We also discuss strategies to apply the CRISPR/Cas9 system to generating targeted genome modifications in whole plants.

Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing is a ground-breaking biotechnology for agricultural applications such as precision breeding in crop plants. Agrobacterium-mediated CRISPR delivery has been successfully adapted for gene knockout applications for basic research and agricultural technology development. However, selecting proper single-guide RNA (sgRNA) for CRISPR binary constructs to induce double-strand break in certain target genes has presented difficulties mainly due to unpredictable in vivo sgRNA activities. Therefore, more than three independent CRISPR constructs, each harboring different sgRNAs, are often applied to ensure the desired CRISPR-induced knockout alleles. Here, we report a zero-background CRISPR binary vector platform, featuring ccdB conjugation within sgRNA cloning cassette, which is later removed by AarI endonuclease, that allows positive survival selection for bona-fide sgRNA clones and effective exclusion of uncut or self-ligated ‘background’ negative clones. We demonstrate the advantage of using the zero-background CRISPR binary platform in a high-throughput pooled cloning strategy of multiple different sgRNAs which produced Agrobacteria containing multiple sgRNAs without any background. We also tested the integrity of pooled CRISPR sgRNA construct libraries during extended bacterial culture and during the transfer between Escherichia coli to Agrobacterium, and verified that the fidelity of sgRNA species representation was faithfully maintained during library generation.

Research highlights for issue 6: the CRISPR/Cas revolution

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been reported for precise genome modification in many plants. In the current study, we demonstrate a successful mutation in phytoene desaturase (RAS-PDS) of banana cv. Rasthali using the CRISPR/Cas9 system. Two PDS genes were isolated from Rasthali (RAS-PDS1 and RAS-PDS2), and their protein sequence analysis confirmed that both PDS comprises conserved motifs for enzyme activity. Phylogenetic analysis of RAS-PDS1 and RAS-PDS2 revealed a close evolutionary relationship with other monocot species. The tissue-specific expression profile of RAS-PDS1 and RAS-PDS2 in Rasthali suggested differential regulation of the genes. A single 19-bp guide RNA (gRNA) was designed to target the conserved region of these two RAS-PDS and transformed with Cas9 in embryogenic cell suspension (ECS) cultures of cv. Rasthali. Complete albino and variegated phenotype were observed among regenerated plantlets. DNA sequencing of 13 plants confirmed the indels with 59% mutation frequency in RAS-PDS, suggesting activation of the non-homologous end-joining (NHEJ) pathway. The majority of mutations were either insertion (1-5) or deletion (1-4) of nucleotides near to protospacer adjacent motif (PAM). These mutations have created stop codons in RAS-PDS sequences which suggest premature termination of RAS-PDS protein synthesis. The decreased chlorophyll and total carotenoid contents were detected in mutant lines that revealed the functional disruption of both RAS-PDS genes. Our results demonstrate that genome editing through CRISPR/Cas9 can be applied as an efficient tool for banana genome modification.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) act as an adaptive immune system against invading nucleic acids and bacteriophages in bacteria and archaea. Based on the constitution of effector protein, CRISPR/Cas is broadly divided into multiple types and subtypes. Among these, type VI CRISPR/Cas system is of special attention with four subtypes, namely, VI-A, VI-B, VI-C, and VI-D, and are believed to have evolutionary origin from transposons. These subtypes exhibit variations in structural architecture and mechanism and have diverse Cas13a (C2c2), Cas13b1 (C2c6), Cas13b2 (C2c6), Cas13c (C2c7) and Cas13d effector proteins. CRISPR/Cas13 ribonuclease processes pre-crRNA to mature crRNA which targets and knockdown single-stranded RNA of phage genome during viral interference. The high specificity RNA guiding and RNA-targeting capacity of this protein enables to fuse with several effector molecules, opening new avenues in the field of Cas13-mediated RNA targeting, tracking, and editing. CRISPR/Cas13 has a unique feature of targeting RNAs including plants, so it can be used as a new tool for engineering interference against plant pathogens including RNA viruses, with better specificity and for other RNA modifications in plants. Fluorescent probe-tagged deactivated programmable Cas13 proteins could be used as an alternative tool for in vitro RNA studies. The engineered Cas13 can also be used for programmable RNA editing. The high target specificity, low cost, and user-friendly operation of CRISPR/Cas13 make this an effective tool for several RNA-based research studies and applications. Therefore, the focus of this chapter is upon classification of CRISPR/Cas system, structural and functional diversity of type VI CRISPR/Cas system including its discovery and origin, mechanism, and role of Cas13 in RNA editing of plants.