Plant agriculture is poised at a technological inflection point. Recent advances in genome engineering make it possible to precisely alter DNA sequences in living cells, providing unprecedented ...control over a plant's genetic material. Potential future crops derived through genome engineering include those that better withstand pests, that have enhanced nutritional value, and that are able to grow on marginal lands. In many instances, crops with such traits will be created by altering only a few nucleotides among the billions that comprise plant genomes. As such, and with the appropriate regulatory structures in place, crops created through genome engineering might prove to be more acceptable to the public than plants that carry foreign DNA in their genomes. Public perception and the performance of the engineered crop varieties will determine the extent to which this powerful technology contributes towards securing the world's food supply.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Generating and applying new knowledge from the wealth of available genomic information is hindered, in part, by the difficulty of altering nucleotide sequences and expression of genes in living cells ...in a targeted fashion. Progress has been made in engineering DNA binding domains to direct proteins to particular sequences for mutagenesis or manipulation of transcription; however, achieving the requisite specificities has been challenging. Transcription activator—like (TAL) effectors of plant pathogenic bacteria contain a modular DNA binding domain that appears to overcome this challenge. Comprising tandem, polymorphic amino acid repeats that individually specify contiguous nucleotides in DNA, this domain is being deployed in DNA targeting for applications ranging from understanding gene function in model organisms to improving traits in crop plants to treating genetic disorders in people.
Genome editing using sequence-specific nucleases (SSNs) offers an alternative approach to conventional genetic engineering and an opportunity to extend the benefits of genetic engineering in ...agriculture. Currently available SSN platforms, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)) have been used in a range of plant species for targeted mutagenesis via non-homologous end joining (NHEJ) are just beginning to be explored in crops such as potato (Solanum tuberosum Group Tuberosum L.). In this study, CRISPR/Cas reagents expressing one of two single-guide RNA (sgRNA) targeting the potato ACETOLACTATE SYNTHASE1 (StALS1) gene were tested for inducing targeted mutations in callus and stable events of diploid and tetraploid potato using Agrobacterium-mediated transformation with either a conventional T-DNA or a modified geminivirus T-DNA. The percentage of primary events with targeted mutations ranged from 3-60% per transformation and from 0-29% above an expected threshold based on the number of ALS alleles. Primary events with targeted mutation frequencies above the expected threshold were used for mutation cloning and inheritance studies using clonal propagation and crosses or selfing. Four of the nine primary events used for mutation cloning had more than one mutation type, and eight primary events contained targeted mutations that were maintained across clonal generations. Somatic mutations were most evident in the diploid background with three of the four primary events having more than two mutation types at a single ALS locus. Conversely, in the tetraploid background, four of the five candidates carried only one mutation type. Single targeted mutations were inherited through the germline of both diploid and tetraploid primary events with transmission percentages ranging from 87-100%. This demonstration of CRISPR/Cas in potato extends the range of plant species modified using CRISPR/Cas and provides a framework for future studies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Crop improvement relies heavily on genetic variation that arises spontaneously through mutation. Modern breeding methods are very adept at combining this genetic variation in ways that achieve ...remarkable improvements in plant performance. Novel traits have also been created through mutation breeding and transgenesis. The advent of gene editing, however, marks a turning point: With gene editing, synthetic variation will increasingly supplement and, in some cases, supplant the genetic variation that occurs naturally. We are still in the very early stages of realizing the opportunity provided by plant gene editing. At present, typically only one or a few genes are targeted for mutation at a time, and most mutations result in loss of gene function. New technological developments, however, promise to make it possible to perform gene editing at scale. RNA virus vectors, for example, can deliver gene-editing reagents to the germ line through infection and create hundreds to thousands of diverse mutations in the progeny of infected plants. With developmental regulators, edited somatic cells can be induced to form meristems that yield seed-producing shoots, thereby increasing throughput and shrinking timescales for creating edited plants. As these approaches are refined and others developed, they will allow for accelerated breeding, the domestication of orphan crops and the reengineering of metabolism in a more directed manner than has ever previously been possible.
Highlights • Rewriting genomes will play an important role in plant synthetic biology. • Sequence-specific nucleases enable almost any DNA sequence change in plant cells. • The advantages and ...limitations of current sequence-specific nucleases are discussed. • A comprehensive list of recent plant genome engineering achievements is provided. • Achievements in genome engineering are related to plant synthetic biology projects.
Plant gene editing is typically performed by delivering reagents such as Cas9 and single guide RNAs to explants in culture. Edited cells are then induced to differentiate into whole plants by ...exposure to various hormones. The creation of edited plants through tissue culture is often inefficient, time-consuming, works for only limited species and genotypes, and causes unintended changes to the genome and epigenome. Here we report two methods to generate gene-edited dicotyledonous plants through de novo meristem induction. Developmental regulators and gene-editing reagents are delivered to somatic cells of whole plants. This induces meristems that produce shoots with targeted DNA modifications, and gene edits are transmitted to the next generation. The de novo induction of gene-edited meristems sidesteps the need for tissue culture and promises to overcome a bottleneck in plant gene editing.
The Streptococcus pyogenes CRISPR-Cas9 system effectively mediates RNA-guided DNA double-strand breaks and is used for genome editing in many different organisms, including plants （Puchta, 2016）. ...CRISPR-Cas9 is a two-component system in which the Cas9 protein is expressed from a Pol II promoter （Lowder et al., 2015）. In contrast, the sgRNAs are typically expressed from Pol III promoters, such as U6 and U3.
Breeding of crops over millennia for yield and productivity has led to reduced genetic diversity. As a result, beneficial traits of wild species, such as disease resistance and stress tolerance, have ...been lost. We devised a CRISPR-Cas9 genome engineering strategy to combine agronomically desirable traits with useful traits present in wild lines. We report that editing of six loci that are important for yield and productivity in present-day tomato crop lines enabled de novo domestication of wild Solanum pimpinellifolium. Engineered S. pimpinellifolium morphology was altered, together with the size, number and nutritional value of the fruits. Compared with the wild parent, our engineered lines have a threefold increase in fruit size and a tenfold increase in fruit number. Notably, fruit lycopene accumulation is improved by 500% compared with the widely cultivated S. lycopersicum. Our results pave the way for molecular breeding programs to exploit the genetic diversity present in wild plants.
Agriculture has reached a technological inflection point. The development of novel gene editing tools and methods for their delivery to plant cells promises to increase genome malleability and ...transform plant biology. Whereas gene editing is capable of making a myriad of DNA sequence modifications, its widespread adoption has been hindered by a number of factors, particularly inefficiencies in creating precise DNA sequence modifications and ineffective methods for delivering gene editing reagents to plant cells. Here, we briefly overview the principles of plant genome editing and highlight a subset of the most recent advances that promise to overcome current limitations.
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