Sukumar Biswas

Bio: Sukumar Biswas is an academic researcher from Shanghai Jiao Tong University. The author has contributed to research in topics: CRISPR & Cas9. The author has an hindex of 5, co-authored 11 publications receiving 74 citations. Previous affiliations of Sukumar Biswas include University of Adelaide & Islamic University.

CRISPR/Cas systems: opportunities and challenges for crop breeding

Abstract: 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.

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

Abstract: 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.

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

Abstract: 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 single‑stranded 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.

Integrative Genomics Viewer

Abstract: Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Mutagenesis in Rice: The Basis for Breeding a New Super Plant.

Abstract: The high selection pressure applied in rice breeding since its domestication thousands of years ago has caused a narrowing in its genetic variability. Obtaining new rice cultivars therefore becomes a major challenge for breeders and developing strategies to increase the genetic variability has demanded the attention of several research groups. Understanding mutations and their applications have paved the way for advances in the elucidation of a genetic, physiological, and biochemical basis of rice traits. Creating variability through mutations has therefore grown to be among the most important tools to improve rice. The small genome size of rice has enabled a faster release of higher quality sequence drafts as compared to other crops. The move from structural to functional genomics is possible due to an array of mutant databases, highlighting mutagenesis as an important player in this progress. Furthermore, due to the synteny among the Poaceae, other grasses can also benefit from these findings. Successful gene modifications have been obtained by random and targeted mutations. Furthermore, following mutation induction pathways, techniques have been applied to identify mutations and the molecular control of DNA damage repair mechanisms in the rice genome. This review highlights findings in generating rice genome resources showing strategies applied for variability increasing, detection and genetic mechanisms of DNA damage repair.

Comparison of CRISPR-Cas9/Cas12a Ribonucleoprotein Complexes for Genome Editing Efficiency in the Rice Phytoene Desaturase (OsPDS) Gene.

Abstract: Delivery of CRISPR reagents into cells as ribonucleoprotein (RNP) complexes enables transient editing, and avoids CRISPR reagent integration in the genomes. Another technical advantage is that RNP delivery can bypass the need of cloning and vector construction steps. In this work we compared efficacies and types of edits for three Cas9 (WT Cas9 nuclease, HiFi Cas9 nuclease, Cas9 D10A nickase) and two Cas12a nucleases (AsCas12a and LbCas12a), using the rice phytoene desaturase (PDS) gene as a target site. Delivery of two Cas9 nucleases (WT Cas9, and HiFi Cas9) and one Cas12a nuclease (LbCas12a) resulted in targeted mutagenesis of the PDS gene. LbCas12a had a higher editing efficiency than that of WT Cas9 and HiFi Cas9. Editing by Cas9 enzymes resulted in indels (1–2 bp) or larger deletions between 20-bp to 30-bp, which included the loss of the PAM site; whereas LbCas12a editing resulted in deletions ranging between 2 bp to 20 bp without the loss of the PAM site. In this work, when a single target site of the rice gene OsPDS was evaluated, the LbCas12a RNP complex achieved a higher targeted mutagenesis frequency than the AsCas12a or Cas9 RNPs.