Genome Editing in Potato with CRISPR/Cas9 Nadakuduti, Satya Swathi; Starker, Colby G; Voytas, Daniel F ...
Methods in molecular biology (Clifton, N.J.),
2019, Letnik:
1917
Journal Article
Cultivated potato, Solanum tuberosum Group Tuberosum L. (2n = 4x = 48) is a heterozygous tetraploid crop that is clonally propagated, thereby resulting in identical genotypes. Due to the lack of ...sexual reproduction and its concomitant segregation of alleles, genetic engineering is an efficient way of introducing crop improvement traits in potato. In recent years, genome-editing via the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system for targeted genome modifications has emerged as the most powerful method due to the ease in designing and construction of gene-specific single guide RNA (sgRNA) vectors. These sgRNA vectors are easily reprogrammable to direct Streptococcus pyogenes Cas9 (SpCas9) to generate double stranded breaks (DSBs) in the target genomes that are then repaired by the cell via the error-prone non-homologous end-joining (NHEJ) pathway or by precise homologous recombination (HR) pathway. CRISPR/Cas9 technology has been successfully implemented in potato for targeted mutagenesis to generate knockout mutations (by means of NHEJ) as well as gene targeting to edit an endogenous gene (by HR). In this chapter, we describe procedures for designing sgRNAs, protocols to clone sgRNAs for CRISPR/Cas9 constructs to generate knockouts, design of donor repair templates and use geminivirus replicons (GVRs) to facilitate gene-editing by HR in potato. We also describe tissue culture procedures in potato for Agrobacterium-mediated transformation to generate gene-edited events along with their molecular characterization.
The
CRISPR system is composed of a Cas9 endonuclease (
Cas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically,
Cas9 proteins could cleave as many targeted ...loci as gRNAs bind in a genome.
We introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a
Cas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (
) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by
Cas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.
This multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.
CRISPR-Cas resonates a revolutionary genome editing technology applicable through a horizon spreading across microbial organism to higher plant and animal. This technology can be harnessed with ease ...to understand the basic genetics of a living system by altering sequence of individual genes and characterizing their functions. The precision of this technology is unparallel. It allows very precise and targeted base pair level edits in the genome. Here, in the current chapter, we have provided end-to-end process outline on how to generate genome edited plants in crops like rice to evaluate for agronomic traits associated with yield, disease resistance and abiotic stress tolerance, etc. Genome editing process includes designing of gene editing strategy, vector construction, plant transformation, molecular screening, and phenotyping under control environment conditions. Furthermore, its application for development of commercial crop product may require additional processes, including field trials in the target geography for evaluation of product efficacy. Evaluation of genome edited lines in controlled greenhouse/net house or open field condition requires few generations for outcrossing with wild-type parent to eliminate and/or reduce any potential pleiotropic effect in the edited genome which may arise during the process. The genome edited plant selected for advancement shall harbor the genome with only the intended changes, which can be analyzed by various molecular techniques, advanced sequencing methods, and genomic data analysis tools. CRISPR-Cas-based genome editing has opened a plethora of opportunities in agriculture as well as human health.
The identification of risks associated with novel agricultural products of plant origin obtained via genome editing is an important aspect of genetic engineering. An extensive discussion is currently ...ongoing worldwide to clarify the similarities and differences between the “old” risks of “classic” GM plants and the “new” ones associated with genome editing, the lack of existing methods for identification and assessment of new risks. We propose here the concept of “safe by design” as applied to protection that is a new interesting tool that introduces good known standards of safety into plant bioengineering. This approach states that design options are identified to minimize or prevent risks and off-target of genome editing at the concept stage. The correlation between experimentally determined and in silico predicted off-target gRNA activity is a major challenge in the CRISPR system application. Today the most studies are focused on efficiency of gRNA design, while we pay attention specifically to the bioinformatics search and study of potential promoters, as the potential risk associates with a possible unplanned change in the transcriptional activity of promoters. We conveyed these strategies in the form of a risk assessment framework for regulation of new genetic technologies.
CRISPR/Cas9 is a technology evolved from modified type II immune system of bacteria and archaea. Exploitation of this bacterial immune system in all eukaryotes including plants may lead to ...site-specific targeted genome engineering. Genome engineering is objectively utilized to express/silence a trait harbouring gene in the plant genome. In this review, different genetic engineering techniques including classical breeding, RNAi and genetic transformation and synthetic sequence-specific nucleases (zinc finger nucleases; ZFNs and transcription activator-like effector nuclease; TALENs) techniques have been described and compared with advanced genome editing technique CRISPR/Cas9, on the basis of their merits and drawbacks. This revolutionary genome engineering technology has edge over all other approaches because of its simplicity, stability, specificity of the target and multiple genes can be engineered at a time. CRISPR/Cas9 requires only Cas9 endonuclease and single guide RNA, which are directly delivered into plant cells via either vector-mediated stable transformation or transient delivery of ribonucleoproteins (RNPs) and generate double-strand breaks (DSBs) at target site. These DSBs are further repaired by cell endogenous repairing pathways via HDR or NHEJ. The major advantage of CRISPR/Cas9 system is that engineered plants are considered Non-GM; can be achieved using in vitro expressed RNPs transient delivery. Different variants of Cas9 genes cloned in different plasmid vectors can be used to achieve different objectives of genome editing including double-stranded DNA break, single-stranded break, activate/repress the gene expression. Fusion of Cas9 with fluorescent protein can lead to visualize the expression of the CRISPR/Cas9 system. The applications of this technology in plant genome editing to improve different plant traits are comprehensively described.
Multiplex CRISPR-Cas9 nuclease mediated genome editing is an efficient method for disrupting gene function in plants. Use of CRISPR-Cas9 has escalated rapidly in recent years and is expected to ...become routine practice in molecular biology and related fields of research. Due to the relatively novel and widespread adoption of this technology, first-time users may not have regular access to experienced guidance or technical support from peers or mentors. Here, we offer guidance and technical support in the form of a detailed and tested protocol for simultaneous targeting of three separate loci on the TRANSPARENT TESTA 4 (TT4) gene in Arabidopsis thaliana using multiplex CRISPR-Cas9. Although we target multiple loci on a single gene in Arabidopsis, the same approach can be used to target multiple genes or alleles in other plant species as well. We recommend the use of a molecular toolkit to streamline the process and make recommendations for this type of approach. The protocol starts with an overview of the reagents and covers designing of gRNAs and assembly of components into a final T-DNA delivery molecule through Golden Gate cloning and Multisite Gateway LR recombination.
The tribe Triticeae includes important agricultural crops, such as bread wheat, durum wheat, barley, rye, and triticale. Research in the field of reverse genetics and genetic engineering of Triticeae ...received a new impetus as the CRISPR/Cas genome editing system came into broad use. The review describes and analyzes the data on recent advances in genomic editing of cultivated plants of the tribe Triticeae and tools used in the field. The tools most commonly used for genome editing in Triticeae include the codon-optimized
Cas9
gene under the control of the maize ubiquitin gene promoter and guide RNAs under the control of Pol III promoters U6 and U3 in one or more binary vectors. Phosphinothricin and hygromycin resistance genes are used as selectable genes. Agrobacterium-mediated transformation and biolistics are performed to obtain genome-edited plants, and immature embryos are used as explants. Approaches developed to overcome the problem of low regenerative capacity of Triticeae include
in planta
transformation of shoot apical meristems, transformation of microspores and pollen grains, and the use of haploinductors. Bread wheat and barley were subject to genomic editing in the majority of studies published to date, and durum wheat and triticale were recently used in CRISPR/Cas knockout studies of target genes. Further progress in the development of genome editing of cultivated plants of the tribe Triticeae should be aimed at expanding the range of species and varieties involved and overcoming the problems of low regenerative capacity. This will allow genetic modification of elite varieties, which will be in demand in agricultural production.
The synthesis of secondary metabolites plays a central role in the survival of plants and their resistance to biotic and abiotic stress. Nevertheless, fundamental and applied studies of plant ...secondary metabolites, polyisoprenols, remain underdeveloped. The wide distribution of polyisoprenols in plants shows their important role in cellular metabolism. Plant polyisoprenols are synthesized by
cis
-prenyltransferases (CPTs), the study of which is necessary to understand the synthesis pathways, localization, and functions of plant polyisoprenols. Bryophytes, including the liverwort
Marchantia polymorpha
, are a unique group of plants with great potential for the study of CPTs. We analyzed the genome of
M. polymorpha
and identified seven CPT genes, which are homologous to the
AtCPT7
and
AtCPT3
genes of
Arabidopsis thaliana
, involved in the synthesis of polyisoprenols. Four individual lines of
M. polymorpha
plants with mutations in the
MpCPT7.37
gene were obtained. It was shown that in three lines the mutation led to a translational frameshift and gene knockout. However, knockout of only the type 7 CPTs had no effect on plant growth and survival. Analysis of the antibacterial activity of mutant plant tissue extracts did not reveal significant changes compared to wild-type tissue extracts, this could be related to a compensatory effect of the activity of other CPTs. These data give us the required base for the further studies of bryophytes CPTs and their products.
Targeted modification of plant genome is key to elucidating and manipulating gene functions in plant research and biotechnology. The clustered regularly interspaced short palindromic repeats ...(CRISPR)/CRISPR-associated protein (Cas) technology is emerging as a powerful genome-editing method in diverse plants that traditionally lacked facile and versatile tools for targeted genetic engineering. This technology utilizes easily reprogrammable guide RNAs (sgRNAs) to direct Streptococcus pyogenes Cas9 endonuclease to generate DNA double-stranded breaks in targeted genome sequences, which facilitates efficient mutagenesis by error-prone nonhomologous end-joining (NHEJ) or sequence replacement by homology-directed repair (HDR). In this chapter, we describe the procedure to design and evaluate dual sgRNAs for plant codon-optimized Cas9-mediated genome editing using mesophyll protoplasts as model cell systems in Arabidopsis thaliana and Nicotiana benthamiana. We also discuss future directions in sgRNA/Cas9 applications for generating targeted genome modifications and gene regulations in plants.
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.
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