Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes

Abstract Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.

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Conventional and Molecular Techniques from Simple Breeding to Speed Breeding in Crop Plants: Recent Advances and Future Outlook

International Journal of Molecular Sciences ◽

10.3390/ijms21072590 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2590 ◽

Cited By ~ 10

Author(s):

Sunny Ahmar ◽

Rafaqat Ali Gill ◽

Ki-Hong Jung ◽

Aroosha Faheem ◽

Muhammad Uzair Qasim ◽

...

Keyword(s):

Plant Breeding ◽

Genome Editing ◽

Agronomic Traits ◽

Genetic Selection ◽

Crop Plants ◽

Molecular Techniques ◽

Whole Genome Sequence ◽

Crop Breeding ◽

Molecular Approaches ◽

Recent Advances

In most crop breeding programs, the rate of yield increment is insufficient to cope with the increased food demand caused by a rapidly expanding global population. In plant breeding, the development of improved crop varieties is limited by the very long crop duration. Given the many phases of crossing, selection, and testing involved in the production of new plant varieties, it can take one or two decades to create a new cultivar. One possible way of alleviating food scarcity problems and increasing food security is to develop improved plant varieties rapidly. Traditional farming methods practiced since quite some time have decreased the genetic variability of crops. To improve agronomic traits associated with yield, quality, and resistance to biotic and abiotic stresses in crop plants, several conventional and molecular approaches have been used, including genetic selection, mutagenic breeding, somaclonal variations, whole-genome sequence-based approaches, physical maps, and functional genomic tools. However, recent advances in genome editing technology using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated (Cas) proteins have opened the door to a new plant breeding era. Therefore, to increase the efficiency of crop breeding, plant breeders and researchers around the world are using novel strategies such as speed breeding, genome editing tools, and high-throughput phenotyping. In this review, we summarize recent findings on several aspects of crop breeding to describe the evolution of plant breeding practices, from traditional to modern speed breeding combined with genome editing tools, which aim to produce crop generations with desired traits annually.

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Faculty Opinions recommendation of Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727215222.793529809 ◽

2017 ◽

Author(s):

Michael Lassner

Keyword(s):

Genome Editing ◽

Bread Wheat ◽

Ribonucleoprotein Complexes

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Transgene-free Genome Editing in Plants

Frontiers in Genome Editing ◽

10.3389/fgeed.2021.805317 ◽

2021 ◽

Vol 3 ◽

Author(s):

Xiaoyong Gu ◽

Lijing Liu ◽

Huawei Zhang

Keyword(s):

Genome Editing ◽

Genetically Modified Organisms ◽

Crop Improvement ◽

Crop Breeding ◽

Transgene Integration ◽

Advantages And Disadvantages ◽

Genetic Segregation ◽

Cas9 Protein ◽

Dna Vectors ◽

The Impact

Genome editing is widely used across plant species to generate and study the impact of functional mutations in crop improvement. However, transgene integration in plant genomes raises important legislative concerns regarding genetically modified organisms. Several strategies have been developed to remove or prevent the integration of gene editor constructs, which can be divided into three major categories: 1) elimination of transgenic sequences via genetic segregation; 2) transient editor expression from DNA vectors; and 3) DNA-independent editor delivery, including RNA or preassembled Cas9 protein-gRNA ribonucleoproteins (RNPs). Here, we summarize the main strategies employed to date and discuss the advantages and disadvantages of using these different tools. We hope that our work can provide important information concerning the value of alternative genome editing strategies to advance crop breeding.

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CRISPR/Cas9 Genome Editing in Bread Wheat (Triticum aestivum L.) Genetic Improvement

Advances in Plant Breeding Strategies: Cereals ◽

10.1007/978-3-030-23108-8_12 ◽

2019 ◽

pp. 453-469

Author(s):

Soleyman Dayani ◽

Mohammad R. Sabzalian ◽

Maryam Mazaheri-Tirani

Keyword(s):

Triticum Aestivum ◽

Genome Editing ◽

Bread Wheat ◽

Genetic Improvement ◽

Triticum Aestivum L

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Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system

Plant Methods ◽

10.1186/1746-4811-9-39 ◽

2013 ◽

Vol 9 (1) ◽

pp. 39 ◽

Cited By ~ 344

Author(s):

Khaoula Belhaj ◽

Angela Chaparro-Garcia ◽

Sophien Kamoun ◽

Vladimir Nekrasov

Keyword(s):

Genome Editing ◽

Crop Plants ◽

Targeted Mutagenesis ◽

Plant Genome

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AGRICULTURAL PRODUCTS IN MEDIEVAL MOSCOW (BASED ON THE MATERIALS OF ARCHAEOLOGICAL EXCAVATIONS IN THE MOSCOW KREMLIN IN 2016-2018)

Краткие сообщения Института археологии (КСИА) ◽

10.25681/iaras.0130-2620.257.374-392 ◽

2019 ◽

Author(s):

Е.Ю. Лебедева ◽

А.Ю. Сергеев

Keyword(s):

Bread Wheat ◽

Crop Plants ◽

Agricultural Products ◽

Exotic Plant ◽

European Cities ◽

Changes Over Time ◽

The City ◽

Insight Into ◽

Archaeological Excavations ◽

Over Time

В статье представлены результаты археоботанических исследований в Московском Кремле и обсуждается проблема использования растений жителями города с особым акцентом на потреблении зерновой продукции. Материалы рассматриваются по двум хронологическим выборкам (XII - перв. пол. XIII в. и втор. пол. XIII - XV в.), что позволяет проследить динамику изменения археоботанических спектров. Выделяются три специфические черты, характеризующие коллекцию зерновых в Москве. Во-первых, высокая насыщенность зерном культурного слоя во-вторых, стабильно высокий показатель доли ржи на протяжении столетий (ок. 70 ) и, в-третьих, остающийся непонятным факт сокращения на 10 доли овса в поздней выборке. Последнее, по мнению авторов, противоречит логике развития города, требующей увеличения фуражных запасов для лошадей - основного транспортного средства средневековья. Авторы приходят к выводу, что при отсутствии или скудости находок экзотических растений, выступающих маркерами элитного питания в европейских городах, в средневековой Руси в этом качестве могут интерпретироваться обычные зерновые культуры, в частности - мягкая пшеница. The paper presents the results of archaeobotanical studies in the Moscow Kremlin and discusses the use of plants by the city residents with a focus on consumption of crops. The analysis is based on two chronological selections (the 12th - first half of the 13th centuries and the first half of the 13th - 15th centuries) it gives an insight into the changes over time of archaeobotanical spectra. Three specific features characterizing the crop grains in Moscow are singled out. Firstly, abundance of crop plants in the occupation layers secondly, consistently high values of the rye share in total crops throughout centuries (around 70 ) and, thirdly, the reduction in the share of oats by 10 in the later sample for some inexplicable reasons. In the view of the authors, the latter fact contradicts the logical development of the city that required increase in forage reserves for horses which was the main animal for transportation in the medieval times. The authors come to the conclusion that in the absence or scarcity of exotic plant finds used as markers of luxury food in European cities, common grain crops such as bread wheat can be used as elite food indicator in Medieval Russia.

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Efficient genome editing in multiple salmonid cell lines using ribonucleoprotein complexes

10.1101/2020.04.03.022038 ◽

2020 ◽

Author(s):

Remi L. Gratacap ◽

Ye Hwa Jin ◽

Marina Mantsopoulou ◽

Ross D. Houston

Keyword(s):

Rainbow Trout ◽

Atlantic Salmon ◽

Infectious Diseases ◽

Genome Editing ◽

Cell Lines ◽

Chinook Salmon ◽

Model Systems ◽

Fish Cell ◽

Ribonucleoprotein Complexes ◽

Salmonid Species

AbstractInfectious and parasitic diseases have major negative economic and animal welfare impacts on aquaculture of salmonid species. Improved knowledge of the functional basis of host response and genetic resistance to these diseases is key to developing preventative and treatment options. Cell lines provide a valuable model to study infectious diseases in salmonids, and genome editing using CRISPR provides an exciting avenue to evaluate the function of specific genes in those systems. While CRISPR/Cas9 has been successfully performed in a Chinook salmon cell line (CHSE-214), there are no reports to date of editing of cell lines derived from the most commercially relevant salmonid species Atlantic salmon and rainbow trout, which are difficult to transduce and therefore edit using lentivirus-mediated methods. In the current study, a method of genome editing of salmonid cell lines using ribonucleoprotein (RNP) complexes was optimised and tested in the most commonly-used salmonid fish cell lines; Atlantic salmon (SHK-1 and ASK cell lines), rainbow trout (RTG-2) and Chinook salmon (CHSE-214). Electroporation of RNP based on either Cas9 or Cas12a was efficient at targeted editing of all the tested lines (typically > 90 % cells edited), and the choice of enzyme expands the number of potential target sites for editing within the genomes of these species. These optimised protocols will facilitate functional genetic studies in salmonid cell lines, which are widely used as model systems for infectious diseases in aquaculture.

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Ultra-deep sequencing reveals no evidence of oncogenic mutations or enrichment by ex vivo CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells

10.1101/2021.10.27.466166 ◽

2021 ◽

Author(s):

M. Kyle Cromer ◽

Valentin V. Barsan ◽

Erich Jaeger ◽

Mengchi Wang ◽

Jessica P. Hampton ◽

...

Keyword(s):

Genome Editing ◽

Deep Sequencing ◽

Limit Of Detection ◽

Ex Vivo ◽

High Fidelity ◽

Seed Region ◽

Hematopoietic Stem ◽

Oncogenic Mutations ◽

Ex Vivo Culture ◽

Target Activity

As CRISPR-based therapies enter the clinic, evaluation of the safety remains a critical and still active area of study. While whole genome sequencing is an unbiased method for identifying somatic mutations introduced by ex vivo culture and genome editing, this methodology is unable to attain sufficient read depth to detect extremely low frequency events that could result in clonal expansion. As a solution, we utilized an exon capture panel to facilitate ultra-deep sequencing of >500 tumor suppressors and oncogenes most frequently altered in human cancer. We used this panel to investigate whether transient delivery of high-fidelity Cas9 protein targeted to three different loci (using guide RNAs (gRNAs) corresponding to sites at AAVS1, HBB, and ZFPM2) at day 4 and day 10 timepoints post-editing resulted in the introduction or enrichment of oncogenic mutations. In three separate primary human HSPC donors, we identified a mean of 1,488 variants per Cas9 treatment (at <0.07% limit of detection). After filtering to remove germline and/or synonymous changes, a mean of 3.3 variants remained per condition, which were further reduced to six total mutations after removing variants in unedited treatments. Of these, four variants resided at the predicted off-target site in the myelodysplasia-associated EZH2 gene that were subject to ZFPM2 gRNA targeting in Donors 2 and 3 at day 4 and day 10 timepoints. While Donor 1 displayed on-target cleavage at ZFPM2, we found no off-target activity at EZH2. Sanger sequencing revealed a homozygous single nucleotide polymorphism (SNP) at position 14bp distal from the Cas9 protospacer adjacent motif in EZH2 that eliminated any detectable off-target activity. We found no evidence of exonic off-target INDELs with either of the AAVS1 or HBB gRNAs. These findings indicate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP outside the seed region of the gRNA protospacer is sufficient to eliminate Cas9 off-target activity with this method of delivery into primary, repair competent human HSPCs.

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