Global developments of genome editing in agriculture

Genome editing, particularly using of site-directed nucleases such as the CRISPR system, has spread rapidly through the biological sciences. Genome editing in crops could significantly speed up the progress of breeding programs. It could drive the development of traits in new crops and allow improvements in yield and pest resistance, adaptation to climate change, and industrial and pharmaceutical applications. However biofortification is a key challenge to satisfy nutritional needs in vitamins for developing countries and new consumer’s needs for developed countries. China and the USA lead scientific research in crop editing. Nigeria, being headquarters to numerous research consortia, is the most involved country in Africa. Genome editing in animals including pig, cattle, sheep, and carp, has not merely accelerated research but has made possible research that was previously unfeasible. It has been used to increase disease resistance, to make livestock better adapted to farming or environmental conditions, to increase fertility and growth, and to improve animal welfare. The USA, the UK and China are the most involved countries in animal genome editing. Global food production needs to increase as much as 70 per cent to support the growing population. Genome editing could contribute improving the efficiency of food distribution and reducing waste. Depending on the regulatory conditions, genome editing could open up the field to smaller companies and public labs.

     

2017基因组编辑专刊序言

基因组编辑技术,作为一项生物医学领域的革新技术,已经在动物、植物和微生物基因组改造中得到了广泛的应用。以CRISPR/Cas9为主导的基因组编辑技术掀起了基因组编辑的浪潮,在功能基因组学、遗传改良育种、遗传病治疗等研究中展示出其极大的价值与潜力。本专刊报道了基因组编辑技术的总体状况、在相关领域的基础与应用研究、该技术当前存在的优缺点以及未来展望等。     

Bombyx mori kynurenine 3-monooxygenase gene editing and insect molecular breeding using the clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 system

Genome editing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein (Cas)9, a third-generation gene scissors, and molecular breeding at the genome level are attracting considerable attention as future breeding techniques. In the present study, genetic and phenotypic analyses were conducted to examine the molecular breeding of Bombyx mori through CRISPR/Cas9-mediated editing of the kynurenine 3-monooxygenase (KMO) gene. The synthesized guide RNAs (gRNAs) were analyzed using T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA, and Cas9 complexes were microinjected into silkworm embryos. After microinjection, the hatching rate and the incidence of mutation were determined as 18.1% and 60%, respectively. Gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed; however, certain embryos and moths produced through sib-mating had significant differences compared to the wild-type. In successive generations, a distinct phenotypic change was also observed by continuous mating. Thus, although there are limitations in the phenotypic expression in breeding through the induction of deletion mutations, as in the present study, the process is believed to yield successful results within a shorter period compared to traditional breeding and is safer than transgenic technology.     

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.     

Efficient Human Genome Editing Using SaCas9 Ribonucleoprotein Complexes

Genome editing using RNA‐guided nucleases in their ribonucleoprotein (RNP) form represents a promising strategy for gene modification and therapy because they are free of exogenous DNA integration and have reduced toxicity in vivo and ex vivo. However, genome editing by Cas9 nuclease from Staphylococcus aureus (SaCas9) has not been reported in its RNP form, which recognizes a longer protospacer adjacent motif (PAM), 5′‐NNGRRT‐3′, compared with Streptococcus pyogenes Cas9 (SpCas9) of 5′‐NGG‐3′ PAM. Here, SaCas9‐RNP‐mediated genome editing is reported in human cells. The SaCas9‐RNP displayed efficient genome editing activities of enhanced green fluorescent protein (EGFP) coding gene as well as three endogenous genes (OPA1, RS1, and VEGFA). Further, SaCas9‐RNP is successfully implemented to correct a pathogenic RS1 mutation for X‐linked juvenile retinoschisis. It is also shown that off‐target effects triggered by SaCas9‐RNP are undetectable by targeted deep sequencing. Collectively, this study demonstrates the potential of SaCas9‐RNP‐mediated genome editing in human cells, which could facilitate genome‐editing‐based therapy.     

Where are we with unintended effects in genome editing applications from DNA to phenotype: focus on plant applications

Agriculture has benefited from various conventional techniques for plant breeding, including chemical- or radiation-induced mutagenesis, and to some extent from transgenesis. Genome editing techniques are likely to allow straightforward, cost-effective and efficient gene-specific modifications for identified genetic traits associated to agronomic interest. As for previous plant breeding techniques, genome editing techniques need an appraisal for unintended effects. Hence, an evaluation of potential specific risks associated with genome editing must be considered. The Scientific Committee of the High Council for biotechnology (HCB), using a broad theoretical and literature-based approach, identified three categories of points to consider in terms of hazards in health and environment, as compared to conventional breeding: (1) technical unintended effects related to effector persistence as well as risks associated with off-target modifications or other unintended genome modifications, (2) risks arising from the desired trait and its novelty in the plant, and (3) risks associated with the potential modification of plant breeding practices, owing to efficacy and technical ease-of-use of genome editing (acceleration), be it for single traits or for combined modifications (multiplex genome editing). Due to novelty, HCB also envisions the need for specific risk assessment and management.

     

基因组编辑技术及其在昆虫研究中的应用

基因组编辑技术是进行功能基因组研究的重要工具．锌指核酸酶技术（ZFNs）、类转录激活因子核酸酶技术（TALENs）以及CRISPR／Cas技术是近年来发展起来的3种主流基因组编辑技术．这3种基因组编辑技术的原理都是通过在生物基因组特定位点制造DNA断裂损伤,从而激活机体自身的DNA损伤修复机制,在此过程中引发各种变异．ZFNs是最早发展的通用基因组编辑技术,可用以实施定点敲除和定点敲入变异,但ZFNs技术的发展受限于构建难度大、成本高等缺点．TALENs技术在ZFNs基础上发展而来,较ZFNs技术而言,TALENs技术具备构建灵活度高、成本低等优势．不同于ZFNs与TALENs技术,CRISPR／Cas技术具有独特的DNA靶向机制,这种机制使其非常适合进行多位点编辑．目前,3种技术都在多种物种中成功测试,例如小鼠、斑马鱼、果蝇、线虫和家蚕．在后基因组时代,这些新技术工具必将在未来功能基因组研究中发挥重大作用．     

Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR‐associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii.     

Raising Climate-Resilient Crops: Journey From the Conventional Breeding to New Breeding Approaches

BackgroundIn order to meet the demands of the ever-increasing human population, it has become necessary to raise climate-resilient crops. Plant breeding, which involves crossing and selecting superior gene pools, has contributed tremendously towards achieving this goal during the past few decades. The relatively newer methods of crop improvement based on genetic engineering are relatively simple, and targets can be achieved in an expeditious manner. More recently emerged genome editing technique using CRISPR has raised strong hopes among plant scientists for precise integration of valuable traits and removal of undesirable ones.ConclusionGenome editing using Site-Specific Nucleases (SSNs) is a good alternative to the plant breeding and genetic engineering approaches as it can modify the genomes specifically and precisely at the target site in the host genome. Another added advantage of the genome editing approach is the simpler biosafety regulations that have been adopted by many countries for commercialization of the products thus generated. This review provides a critical assessment of the available methods for improving the stress tolerance in crop plants. Special emphasis has been given on genome editing approach in light of the diversity of tools, which are being discovered on an everyday basis and the practical applications of the same. This information will serve as a beginner’s guide to initiate the crop improvement programs as well as giving technical insight to the expert to plan the research strategically to tackle even multigenic traits in crop plants.     

基因编辑技术在斑马鱼中的应用及研究进展

基因编辑技术通过对特定DNA片段的插入、敲除、修饰或替换等,实现对生物体中目标基因的编辑。与早期基因工程技术将遗传物质随机插入宿主基因组中的方式不同的是,基因编辑技术能够定点需要插入的位置,从而实现对生物体基因组特定位点的准确修饰、人为地改造生物体的遗传信息,目前广泛应用于斑马鱼的基因组学、遗传发育和基因功能研究中。其方法包括诱变技术、Tol2转座子、Morpholino、ZFNs、TALEN和CRISPR/Cas系统等。本研究主要介绍了基因编辑技术的作用机理与发展概况。作为一种精准而高效的基因工程方法,基因编辑技术在近年来得到了飞速地发展。它既可以采用对特定基因的靶向突变来研究基因的功能,也可以通过将功能性基因插入并替代缺陷基因而用于某些遗传性疾病的基因治疗。可以肯定的是,基因编辑技术未来将在基础生物学、医学、生物技术等多个领域具有重要的研究价值和应用价值。