Genome Editing in Chlamydomonas reinhardtii Using Cas9-gRNA Ribonucleoprotein Complex: A Step-by-Step Guide.

Genome editing technologies have provided opportunities to manipulate literally any genomic location, opening new avenues for reverse genetics-based improvements. Among them, CRISPR/Cas9 is the most versatile tool for genome editing applications in prokaryotes and eukaryotes. Here, we provide a guide to successfully carry out high-efficiency genome editing in Chlamydomonas reinhardtii using preassembled CRISPR/Cas9-gRNA ribonucleoprotein (RNP) complexes.

Genotyping by sequencing-based linkage map construction and identification of quantitative trait loci for yield-related traits and oil content in Jatropha (Jatropha curcas L.).

BACKGROUND: Jatropha (Jatropha curcas L.) has been considered as a potential bioenergy crop and its genetic improvement is essential for higher seed yield and oil content which has been hampered due to lack of desirable molecular markers. METHODS AND RESULTS: An F2 population was created using an intraspecific cross involving a Central American line RJCA9 and an Asiatic species RJCS-9 to develop a dense genetic map and for Quantitative trait loci (QTL) identification. The genotyping-by-sequencing (GBS) approach was used to genotype the mapping population of 136 F2 individuals along with the two parental lines for classification of the genotypes based on single nucleotide polymorphism (SNPs). NextSeq 2500 sequencing technology provided a total of 517.23 million clean reads, with an average of ~ 3.8 million reads per sample. We analysed 411 SNP markers and developed 11 linkage groups. The total length of the genetic map was 4092.3 cM with an average marker interval of 10.04 cM. We have identified a total of 83 QTLs for various yield and oil content governing traits. The percentage of phenotypic variation (PV) was found to be in the range of 8.81 to 65.31%, and a QTL showed the maximum PV of 65.3% for a total seed number on the 6th linkage group (LG). CONCLUSIONS: The QTLs detected in this study for various phenotypic traits will lay down the path for marker-assisted breeding in the future and cloning of genes that are responsible for phenotypic variation.

CRISPR-based assays for rapid detection of SARS-CoV-2.

COVID-19 pandemic posed an unprecedented threat to global public health and economies. There is no effective treatment of the disease, hence, scaling up testing for rapid diagnosis of SARS-CoV-2 infected patients and quarantine them from healthy individuals is one the best strategies to curb the pandemic. Establishing globally accepted easy-to-access diagnostic tests is extremely important to understanding the epidemiology of the present pandemic. While nucleic acid based tests are considered to be more sensitive with respect to serological tests but present gold standard qRT-PCR-based assays possess limitations such as low sample throughput, requirement for sophisticated reagents and instrumentation. To overcome these shortcomings, recent efforts of incorporating LAMP-based isothermal detection, and minimizing the number of reagents required are on rise. CRISPR based novel techniques, when merge with isothermal and allied technologies, promises to provide sensitive and rapid detection of SARS-CoV-2 nucleic acids. Here, we discuss and present compilation of state-of-the-art detection techniques for COVID-19 using CRISPR technology which has tremendous potential to transform diagnostics and epidemiology.

CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement.

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA, targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

Development of genome wide transposable elements based repeat junction markers in Jatropha (Jatropha curcas L.).

Jatropha curcas is a potential biodiesel crop and a highly adaptable species to various agro-climatic conditions. In this study, we have utilized transposable elements' (TE) repeat junctions (RJs) which are an important constituent of the genome, used to form a genome-wide molecular marker platform owing to its use in genomic studies of plants. We screened our previously generated Jatropha hybrid genome assembly of size 265 Mbp using RJPrimers pipeline software and identified a total of 1274 TE junctions. For the predicted RJs, we designed 2868 polymerase chain reaction (PCR) based RJ markers (RJMs) flanking the junction regions. In addition to marker design, the identified RJs were utilized to detect 225,517 TEs across the genome. The different types of transposable repeat elements mainly were scattered into Retro, LTR, Copia and Gypsy categories. The efficacy of the designed markers was tested by utilizing a subset of RJMs selected randomly. We have validated 96 randomly selected RJ primers in a group of 32 J. curcas genotypes and more than 90% of the markers effectively intensified as amplicons. Of these, 10 primers were shown to be polymorphic in estimating genetic diversity among the 32 Jatropha lines. UPGMA cluster analysis revealed the formation of two clusters such as A and B exhibiting 85.5% and 87% similarity coefficient respectively. The various RJMs identified in this study could be utilized as a significant asset in Jatropha functional genomics including genome determination, mapping and marker-assisted selection.

Exploitation of Hi-C sequencing for improvement of genome assembly and in-vitro validation of differentially expressing genes in Jatropha curcas L.

Jatropha curcas is one of the major sources of renewable energy due to potential use of its oil as a biofuel. The genome of this crop is constituted by the high content of repetitive elements. We employed the Hi-C proximity ligation technique to re-scaffold our existing hybrid genome assembly of an elite genotype (RJC1) developed using Illumina and Pacbio technologies. We assembled 99.81% of non-truncated reads to achieve 266.80 Mbp of the genome with an N50 value of 1.58 Mb. Furthermore, we compared the efficiency of Hi-C-augmented genome assembly with the hybrid genome assembly and observed a ~ 50% reduction in scaffolds and a tenfold increase in the N50 value. The gene ontology analysis revealed the identification of terms for molecular function (45.52%), cellular component (33.47%), and biological function (20.99%). Comparative genomic analysis of 13-plant species showed the conservation of 414 lipid metabolizing genes identified in the KEGG pathway analysis. Differential gene expression (DGE) studies were conducted in the healthy and Jatropha mosaic virus-infected leaves via RNA-seq analysis and observed gene expression changes for 2185 genes. Out of these, we observed 546 genes having more than two-fold change of transcript level and among these 259 genes were down-regulated and 287 genes were up-regulated. To validate RNA-seq data, two DEGs were selected for gene expression analysis using qRT-PCR and the data was in correlation with in silico results. RNA-seq analysis further shows the identification of some of the candidate genes and may be useful to develop JMV resistant plants after functional validation. This Hi-C genome assembly provides a detailed accurate reference genome which could be utilized to improve Jatropha and other economically important Euphorbiaceae family members.

De Novo Sequencing and Hybrid Assembly of the Biofuel Crop Jatropha curcas L.: Identification of Quantitative Trait Loci for Geminivirus Resistance.

Jatropha curcas is an important perennial, drought tolerant plant that has been identified as a potential biodiesel crop. We report here the hybrid de novo genome assembly of J. curcas generated using Illumina and PacBio sequencing technologies, and identification of quantitative loci for Jatropha Mosaic Virus (JMV) resistance. In this study, we generated scaffolds of 265.7 Mbp in length, which correspond to 84.8% of the gene space, using Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis. Additionally, 96.4% of predicted protein-coding genes were captured in RNA sequencing data, which reconfirms the accuracy of the assembled genome. The genome was utilized to identify 12,103 dinucleotide simple sequence repeat (SSR) markers, which were exploited in genetic diversity analysis to identify genetically distinct lines. A total of 207 polymorphic SSR markers were employed to construct a genetic linkage map for JMV resistance, using an interspecific F2 mapping population involving susceptible J. curcas and resistant Jatropha integerrima as parents. Quantitative trait locus (QTL) analysis led to the identification of three minor QTLs for JMV resistance, and the same has been validated in an alternate F2 mapping population. These validated QTLs were utilized in marker-assisted breeding for JMV resistance. Comparative genomics of oil-producing genes across selected oil producing species revealed 27 conserved genes and 2986 orthologous protein clusters in Jatropha. This reference genome assembly gives an insight into the understanding of the complex genetic structure of Jatropha, and serves as source for the development of agronomically improved virus-resistant and oil-producing lines.