CRISPR/Cas systems: opportunities and challenges for crop breeding.

Increasing crop production to meet the demands of a growing population depends largely on crop improvement through new plant-breeding techniques (NPBT) such as genome editing. CRISPR/Cas systems are NPBTs that enable efficient target-specific gene editing in crops, which is supposed to accelerate crop breeding in a way that is different from genetically modified (GM) technology. Herein, we review the applications of CRISPR/Cas systems in crop breeding focusing on crop domestication, heterosis, haploid induction, and synthetic biology, and summarize the screening methods of CRISPR/Cas-induced mutations in crops. We highlight the importance of molecular characterization of CRISPR/Cas-edited crops, and pay special attentions to emerging highly specific genome-editing tools such as base editors and prime editors. We also discuss future improvements of CRISPR/Cas systems for crop improvement.

Effective identification of CRISPR/Cas9-induced and naturally occurred mutations in rice using a multiplex ligation-dependent probe amplification-based method.

KEY MESSAGE: A multiplex ligation-dependent probe amplification (MLPA)-based method was developed and successfully utilized to efficiently detect both CRISPR/Cas9-induced and naturally occurred mutations in rice. The site-specific nuclease-based CRISPR/Cas9 system has emerged as one of the most efficient genome editing tools to modify multiple genomic targets simultaneously in various organisms, including plants for both fundamental and applied researches. Screening for both on-target and off-target mutations in CRISPR/Cas9-generated mutants at the early stages is an indispensable step for functional analysis and subsequent application. Various methods have been developed to detect CRISPR/Cas9-induced mutations in plants. Still, very few have focused on the detection of both on- and off-targets simultaneously, let alone the detection of natural mutations. Here, we report a multiplex capable method that allows to detect CRISPR/Cas9 induced on- and off-target mutations as well as naturally occurred mutation based on a multiplex ligation-dependent probe amplification (MLPA) method. We demonstrated that unlike other methods, the modified target-specific MLPA method can accurately identify any INDELs generated naturally or by the CRISPR/Cas9 system and that it can detect natural variation and zygosity of the CRISPR/Cas9-generated mutants in rice as well. Furthermore, its high sensitivity allowed to define INDELs down to 1 bp and substitutions to a single nucleotide. Therefore, this sensitive, reliable, and cheap method would further accelerate functional analysis and marker-assisted breeding in plants, including rice.

Development of methods for effective identification of CRISPR/Cas9-induced indels in rice.

KEY MESSAGE: Two methods, PCR and amplicon labeling based, were developed and successfully applied to reliably detect CRISPR/Cas9 induced indels in rice. The use of CRISPR/Cas9 has emerged as a powerful nuclease-based genome editing tool in several model organisms including plants for mutagenesis by inducing precise gene editing through efficient double strand DNA breaks (DSBs) at the target site and subsequent error-prone non-homologous end joining (NHEJ) repair, leading to indel mutations. Different molecular methods including enzymatic mismatch cleavage (EMC), high-resolution melting curve analysis (HRMA) and conventional polymerase chain reaction (PCR) combined with ligation detection reaction (LDR) have been developed to quick identify CRISPR/Cas9 induced mutations. However, their intrinsic drawbacks limit their application in the identification of indel mutants in plants. Here we present two methods (one simple PCR based and the other amplicon labeling based) for effective and sensitive detection of CRISPR/Cas9 induced indels in rice. In PCR-based method, targets were amplified using two pairs of primers for each target locus and visualized on gel electrophoresis, while in amplicon labeling-based method, targets were amplified using tri-primers (with one a universal 6-FAM 5'-labelled) and detected by DNA capillary electrophoresis. Both methods can accurately define indel sizes down to ± 1 bp, and are amenable for high throughput analysis, therefore, will significantly facilitate the identification of indel mutants generated by CRISPR/Cas9 for further functional analysis and breeding in rice and other plants.

Investigation of CRISPR/Cas9-induced SD1 rice mutants highlights the importance of molecular characterization in plant molecular breeding.

Although Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has been widely used for basic research in model plants, its application for applied breeding in crops has faced strong regulatory obstacles, due mainly to a poor understanding of the authentic output of this system, particularly in higher generations. In this study, different from any previous studies, we investigated in detail the molecular characteristics and production performance of CRISPR/Cas9-generated SD1 (semi-dwarf 1) mutants from T2 to T4 generations, of which the selection of T1 and T2 was done only by visual phenotyping for semidwarf plants. Our data revealed not only on- and off-target mutations with small or lager indels but also exogenous elements in T2 plants. All indel mutants passed stably to T3 or T4 without additional modifications independent on the presence of Cas9, while some lines displayed unexpected hereditary patterns of Cas9 or some exogenous elements. In addition, effects of various SD1 alleles on rice height and yield differed depending on genetic backgrounds. Taken together, our data showed that the CRISPR/Cas9 system is effective in producing homozygous mutants for functional analysis, but it may be not as precise as expected in rice, and that early and accurate molecular characterization and screening must be carried out for generations before transitioning of the CRISPR/Cas9 system from laboratory to field.

A Novel RNA Virus, Macrobrachium rosenbergii Golda Virus (MrGV), Linked to Mass Mortalities of the Larval Giant Freshwater Prawn in Bangladesh.

Mass mortalities of the larval stage of the giant freshwater prawn, Macrobrachium rosenbergii, have been occurring in Bangladesh since 2011. Mortalities can reach 100% and have resulted in an 80% decline in the number of hatcheries actively producing M. rosenbergii. To investigate a causative agent for the mortalities, a disease challenge was carried out using infected material from a hatchery experiencing mortalities. Moribund larvae from the challenge were prepared for metatranscriptomic sequencing. De novo virus assembly revealed a 29 kb single­stranded positive-sense RNA virus with similarities in key protein motif sequences to yellow head virus (YHV), an RNA virus that causes mass mortalities in marine shrimp aquaculture, and other viruses in the Nidovirales order. Primers were designed against the novel virus and used to screen cDNA from larvae sampled from hatcheries in the South of Bangladesh from two consecutive years. Larvae from all hatcheries screened from both years were positive by PCR for the novel virus, including larvae from a hatchery that at the point of sampling appeared healthy, but later experienced mortalities. These screens suggest that the virus is widespread in M. rosenbergii hatchery culture in southern Bangladesh, and that early detection of the virus can be achieved by PCR. The hypothesised protein motifs of Macrobrachium rosenbergii golda virus (MrGV) suggest that it is likely to be a new species within the Nidovirales order. Biosecurity measures should be taken in order to mitigate global spread through the movement of post-larvae within and between countries, which has previously been linked to other virus outbreaks in crustacean aquaculture.

Molecular characterization of genetically-modified crops: Challenges and strategies.

Molecular characterization lays a foundation for safety assessment and subsequent monitoring of genetically modified (GM) crops. Due to the target-specific nature, conventional polymerase chain reaction (PCR)-based methods cannot comprehensively detect unintended gene insertions, let alone unknown GM events. As more and more new developed GM crops including new plant breeding technology (NPBT) generated crops are in the pipeline for commercialization, alternative -omics approaches, particularly next generation sequencing, have been developed for molecular characterization of authorized or unauthorized GM (UGM) crops. This review summarizes first those methods, addresses their challenges, and discusses possible strategies for molecular characterization of engineered crops generated by NPBT, highlighting needs for a global information-sharing database and cost-effective, accurate and comprehensive molecular characterization approaches.

The Development of DNA Based Methods for the Reliable and Efficient Identification of Nicotiana tabacum in Tobacco and Its Derived Products.

Reliable methods are needed to detect the presence of tobacco components in tobacco products to effectively control smuggling and classify tariff and excise in tobacco industry to control illegal tobacco trade. In this study, two sensitive and specific DNA based methods, one quantitative real-time PCR (qPCR) assay and the other loop-mediated isothermal amplification (LAMP) assay, were developed for the reliable and efficient detection of the presence of tobacco (Nicotiana tabacum) in various tobacco samples and commodities. Both assays targeted the same sequence of the uridine 5'-monophosphate synthase (UMPS), and their specificities and sensitivities were determined with various plant materials. Both qPCR and LAMP methods were reliable and accurate in the rapid detection of tobacco components in various practical samples, including customs samples, reconstituted tobacco samples, and locally purchased cigarettes, showing high potential for their application in tobacco identification, particularly in the special cases where the morphology or chemical compositions of tobacco have been disrupted. Therefore, combining both methods would facilitate not only the detection of tobacco smuggling control, but also the detection of tariff classification and of excise.