DNA Insert Research Articles

A recombineering system for Bacillus subtilis based on the native phage recombinase pair YqaJ/YqaK

Bacillus subtilis plays an important role in fundamental and applied research, and it has been widely used as a cell factory for the production of enzymes, antimicrobial materials, and chemicals for agriculture, medicine, and industry. However, genetic manipulation tools for B. subtilis have low efficiency. In this work, our goal was to develop a simple recombineering system for B. subtilis. We showed that genome editing can be achieved in B. subtiliis 1A751 through co-expression of YqaJ/YqaK, a native phage recombinase pair found in B. subtilis 168, and the competence master regulator ComK using a double-stranded DNA substrate with short homology arms (100 bp) and a phosphorothioate modification at the 5′-end. Efficient gene knockouts and large DNA insertions were achieved using this new recombineering system in B. subtilis 1A751. As far as we know, this is the first recombineering system using the native phage recombinase pair YqaJ/YqaK in B. subtilis. In conclusion, this new recombineering system provides a simple and fast tool for genetic manipulation of B. subtilis, and it will promote studies of genome function, construction of production strains, and genome mining in B. subtilis.

Deep amplicon sequencing reveals extensive allelic diversity in the erg11/CYP51 promoter and allows multi-population DMI fungicide resistance monitoring in the canola pathogen Leptosphaeria maculans

Continued use of fungicides provides a strong selection pressure towards strains with mutations to render these chemicals less effective. Previous research has shown that resistance to the demethylation inhibitor (DMI) fungicides, which target ergosterol synthesis, in the canola pathogen Leptosphaeria maculans has emerged in Australia and Europe. The change in fungicide sensitivity of individual isolates was found to be due to DNA insertions into the promoter of the erg11/CYP51 DMI target gene. Whether or not these were the only types of mutations and how prevalent they were in Australian populations was explored in the current study. New isolates with reduced DMI sensitivity were obtained from screens on DMI-treated plants, revealing eight independent insertions in the erg11 promoter. A novel deep amplicon sequencing approach applied to populations of ascospores fired from stubble identified an additional undetected insertion allele and quantified the frequencies of all known insertions, suggesting that, at least in the samples processed, the combined frequency of resistant alleles is between 0.0376% and 32.6%. Combined insertion allele frequencies positively correlated with population-level measures of in planta resistance to four different DMI treatments. Additionally, there was no evidence for erg11 coding mutations playing a role in conferring resistance in Australian populations. This research provides a key method for assessing fungicide resistance frequency in stubble-borne populations of plant pathogens and a baseline from which additional surveillance can be conducted in L. maculans. Whether or not the observed resistance allele frequencies are associated with loss of effective disease control in the field remains to be established.

Oncomine Dx Express CE-IVD liquid biopsy assay for non-small cell lung cancer: Performance review and analytical validation.

e21218 Background: Clinical diagnostics assays for oncology are becoming more readily available due to the advancement and democratization of Next-Generation Sequencing (NGS). Additionally, liquid biopsy can be used in NGS to detect genetic variants in circulating tumor DNA and RNA. Liquid biopsy is a less invasive option compared to traditional biopsy methods for early detection and continuous monitoring of cancer treatment outcomes. Here, we discuss screening of over 3,500 non-small cell lung cancer (NSCLC) samples, and the analytical validation of the Oncomine Dx Express Test (ODxET) and Genexus Dx Integrated Sequencer for detection of clinically significant variants in liquid biopsy samples. Methods: Our team screened over 3,500 NSCLC liquid biopsy samples in search of high priority clinical variants. Variants selected for analytical validation studies included ERBB2 exon 20 insertion, EGFR exon 20 insertion, EGFR exon 19 deletion, EGFR T790M, KRAS G12C, BRAF V600E, and RNA fusion isoforms including ALK, NTRK1/2/3, RET, and ROS1 oncogenic drivers. Over 550 screened samples were found to be positive for these variants of interest. This screening was performed on the Genexus Dx Integrated Sequencer according to the user guide. Results: The Genexus Dx Integrated Sequencer automates library preparation, sequencing, analysis, and reporting QC metrics and variant calls. Sequencing run setup is quick and straightforward, taking less than 15 minutes to start a run and just over 24 hours to go from nucleic acid to report. Limit of detection (LoD) for DNA SNVs, insertions, and deletions at 5 ng DNA input level ranged from 0.65% to 1.82% allelic frequency (AF), depending on the variant. The higher DNA input of 30 ng resulted in a lower LoD range, from 0.31% to 0.42% AF. RNA fusion and splice variant LoD at 5 ng sample input ranged from 9.9 to 19.6 molecular counts. The higher 30 ng input resulted in a lower LoD range for RNA variants as well, ranging from 6.4 to 8.0 molecular counts. In the analytical accuracy study, the false positive rate was found to be 0.2% for SNVs, 0% for insertions/deletions, and 0% for fusion targets. In the analytical reproducibility study, the average with-in run repeatability call rate (No Calls excluded) was 99.64% for DNA variants and 98.75% for RNA variants. Conclusions: Here, we demonstrated that the ODxET workflow on Genexus Dx Integrated Sequencer is a fast and efficient tool for testing clinical NSCLC liquid biopsy samples with high sensitivity and specificity.

Ion Channels as Potential Tools for the Diagnosis, Prognosis, and Treatment of HPV-Associated Cancers.

The human papilloma virus (HPV) group comprises approximately 200 genetic types that have a special affinity for epithelial tissues and can vary from producing benign symptoms to developing into complicated pathologies, such as cancer. The HPV replicative cycle affects various cellular and molecular processes, including DNA insertions and methylation and relevant pathways related to pRb and p53, as well as ion channel expression or function. Ion channels are responsible for the flow of ions across cell membranes and play very important roles in human physiology, including the regulation of ion homeostasis, electrical excitability, and cell signaling. However, when ion channel function or expression is altered, the channels can trigger a wide range of channelopathies, including cancer. In consequence, the up- or down-regulation of ion channels in cancer makes them attractive molecular markers for the diagnosis, prognosis, and treatment of the disease. Interestingly, the activity or expression of several ion channels is dysregulated in HPV-associated cancers. Here, we review the status of ion channels and their regulation in HPV-associated cancers and discuss the potential molecular mechanisms involved. Understanding the dynamics of ion channels in these cancers should help to improve early diagnosis, prognosis, and treatment in the benefit of HPV-associated cancer patients.

Suppression of the Arabidopsis cinnamoyl-CoA reductase 1-6 intronic T-DNA mutation by epigenetic modification.

Arabidopsis (Arabidopsis thaliana) transfer DNA (T-DNA) insertion collections are popular resources for fundamental plant research. Cinnamoyl-CoA reductase 1 (CCR1) catalyzes an essential step in the biosynthesis of the cell wall polymer lignin. Accordingly, the intronic T-DNA insertion mutant ccr1-6 has reduced lignin levels and shows a stunted growth phenotype. Here, we report restoration of the ccr1-6 mutant phenotype and CCR1 expression levels after a genetic cross with a UDP-glucosyltransferase 72e1 (ugt72e1),-e2,-e3 T-DNA mutant. We discovered that the phenotypic recovery was not dependent on the UGT72E family loss of function but due to an epigenetic phenomenon called trans T-DNA suppression. Via trans T-DNA suppression, the gene function of an intronic T-DNA mutant was restored after the introduction of an additional T-DNA sharing identical sequences, leading to heterochromatinization and splicing out of the T-DNA-containing intron. Consequently, the suppressed ccr1-6 allele was named epiccr1-6. Long-read sequencing revealed that epiccr1-6, not ccr1-6, carries dense cytosine methylation over the full length of the T-DNA. We showed that the SAIL T-DNA in the UGT72E3 locus could trigger the trans T-DNA suppression of the GABI-Kat T-DNA in the CCR1 locus. Furthermore, we scanned the literature for other potential cases of trans T-DNA suppression in Arabidopsis and found that 22% of the publications matching our query report on double or higher-order T-DNA mutants that meet the minimal requirements for trans T-DNA suppression. These combined observations indicate that intronic T-DNA mutants need to be used with caution since methylation of intronic T-DNA might derepress gene expression and can thereby confound results.

PASTE: a high-throughput method for large DNA insertions

Prime editing (PE) enables precise genome editing at targeted locus without inducing double-stranded breaks (DSBs). Despite its precision, PE lacks the tendency to integrate large DNA fragments into the genome. Recently, Yarnall et al. reported clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 and an integrase-based system that conducts targeted integration of large DNA sequences (~36 kb) into the genome more efficiently.

Precise integration of large DNA sequences in plant genomes using PrimeRoot editors.

A technique for chromosomal insertion of large DNA segments is much needed in plant breeding and synthetic biology to facilitate the introduction of desired agronomic traits and signaling and metabolic pathways. Here we describe PrimeRoot, a genome editing approach to generate targeted precise large DNA insertions in plants. Third-generation PrimeRoot editors employ optimized prime editing guide RNA designs, an enhanced plant prime editor and superior recombinases to enable precise large DNA insertions of up to 11.1 kilobases into plant genomes. We demonstrate the use of PrimeRoot to accurately introduce gene regulatory elements in rice. In this study, we also integrated a gene cassette comprising PigmR, which confers rice blast resistance driven by an Act1 promoter, into a predicted genomic safe harbor site of Kitaake rice and obtain edited plants harboring the expected insertion with an efficiency of 6.3%. We found that these rice plants have increased blast resistance. These results establish PrimeRoot as a promising approach to precisely insert large segments of DNA in plants.

AAV analysis by sedimentation velocity analytical ultracentrifugation: beyond empty and full capsids

The recent surge of therapeutic interest in recombinant adeno-associated viral (AAV) vectors for targeted DNA delivery has brought analytical ultracentrifugation (AUC) into the spotlight. A major concern during formulation of AAV therapeutics is purity of the active species (DNA-containing capsid, or "filled capsids"). Insertion of DNA into AAV is not a highly efficient process; thus, a significant amount of empty and partial/intermediate AAV molecules may exist. Recent guidance from the FDA includes limiting the presence of empty AAV capsids and other impurities to reduce immunotoxicity. While chromatographic techniques (SEC, SEC-MALS, AEX) are often used for empty and full capsid quantitation due to the ease of accessibility and familiarity among most biochemists, the resolution and sensitivity attained by sedimentation velocity (SV-AUC) in the formulation buffer and purification buffers is unmatched. Approaches for using SV-AUC to determine the empty-to-full capsid ratio have already been discussed by others; however, in this report, we focus on the importance of characterizing other impurities, such as free DNA, partially filled capsids, and aggregates that are recognized as species of concern for immunotoxicity. We also demonstrate the usefulness of applying multiple analyses (e.g., c(s), g(s*), WDA) in confirming the presence of and determining the hydrodynamic parameters of these various species.

Abstract 5601: Oncomine™ dx express CE-IVD liquid biopsy assay for non-small cell lung cancer: Performance review and analytical validation

Abstract Introduction - Clinical diagnostics assays for oncology are becoming more readily available due to the advancement and democratization of Next-Generation Sequencing (NGS). Additionally, liquid biopsy can be used in NGS to detect genetic variants in circulating tumor DNA and RNA. Liquid biopsy is a less invasive option compared to traditional biopsy methods for early detection and continuous monitoring of cancer treatment outcomes. Here, we discuss screening of over 3,500 non-small cell lung cancer (NSCLC) samples, and the analytical validation of the Oncomine™ Dx Express Test (ODxET) and Genexus™ Dx Integrated Sequencer for detection of clinically significant variants in liquid biopsy samples. Methods - Our team screened over 3,500 NSCLC liquid biopsy samples in search of high priority clinical variants. Variants selected for analytical validation studies included ERBB2 exon 20 insertion, EGFR exon 20 insertion, EGFR exon 19 deletion, EGFR T790M, KRAS G12C, BRAF V600E, and RNA fusion isoforms including ALK, NTRK1/2/3, RET, and ROS1 oncogenic drivers. Over 550 screened samples were found to be positive for these variants of interest. This screening was performed on the Genexus™ Dx Integrated Sequencer according to the user guide. Results - The Genexus™ Dx Integrated Sequencer automates library preparation, sequencing, analysis, and reporting QC metrics and variant calls. Sequencing run setup is quick and straightforward, taking less than 15 minutes to start a run and just over 24 hours to go from nucleic acid to report. Limit of detection (LoD) for DNA SNVs, insertions, and deletions at 5 ng DNA input level ranged from 0.65% to 1.82% allelic frequency (AF), depending on the variant. The higher DNA input of 30 ng resulted in a lower LoD range, from 0.31% to 0.42% AF. RNA fusion and splice variant LoD at 5 ng sample input ranged from 9.9 to 19.6 molecular counts. The higher 30 ng input resulted in a lower LoD range for RNA variants as well, ranging from 6.4 to 8.0 molecular counts. In the analytical accuracy study, the false positive rate was found to be 0.2% for SNVs, 0% for insertions/deletions, and 0% for fusion targets. In the analytical reproducibility study, the average with-in run repeatability call rate (No Calls excluded) was 99.64% for DNA variants and 98.75% for RNA variants. Conclusion - Here, we demonstrated that the ODxET workflow on Genexus™ Dx Integrated Sequencer is a fast and efficient tool for testing clinical NSCLC liquid biopsy samples with high sensitivity and specificity. For in vitro diagnostic use. Not available in all regions including the United States. Citation Format: Nicholas Siepert, Jeoffrey Schageman, Jian Gu, Thilanka Jayaweera, Madhu Jasti, Luming He, Stephen Wunch, Emilia Ostrowska, Tasha Delacour, Diarra Hassell, Thomas Bowden, Daniela Garcia, Stephanie Tong, Swasti Raut, Nader Ezzedine, Elliott Martinez, Kelli Bramlett. Oncomine™ dx express CE-IVD liquid biopsy assay for non-small cell lung cancer: Performance review and analytical validation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5601.

Anzalone Prime: An Interview with Prime Editing Developer Andrew Anzalone

Anzalone Prime: An Interview with Prime Editing Developer Andrew Anzalone

An artificial positive control for routine detection of rose rosette virus and Phyllocoptes fructiphilus that fit most primers for PCR, LAMP and RPA based assays

AbstractRose (Rosa spp.) is a very important ornamental shrub cultivated worldwide and of value for the pharmaceutical industry. The plant is broadly susceptible to pathogens, including viruses. Rose rosette virus (RRV; virus species Emaravirus rosae) causes multiple symptoms typically rosettes, ultimately leading to death. The virus transmission is by grafting and a wind‐dispersed eriophyoid mite, Phyllocoptes fructiphilus, which survives in winter‐dormant plants. Due to extensive globalization RRV is a threat for the European rose, landscape, nursery and tourism industries. The most common and reliable method used for RRV detection is RT‐PCR. Positive control is indispensable for PCR reliability and can be difficult to obtain for emerging or highly contagious pathogens and are subject to BSL‐2 quarantine. A synthetic artificial positive control (APC) using custom DNA inserts of sense and anti‐sense primers was designed de novo and inserted in a circular plasmid vector to create a positive control for use with most RRV reported primers and eriophyoid mites. This study describes a functional demonstration and development of a rapid, consistent, adaptable and cost‐effective alternative to infected true‐tissue positive control for detection of RRV. The inserted RRV primers are for end point and quantitative RT‐PCR, reverse transcription loop‐mediated isothermal amplification (RT‐LAMP), recombinase‐polymerase amplification (RPA), broad detection of emaravirus and the eriophyoid mite vector Phyllocoptes fructiphilus. The APC‐RRV and RRV infected rose (leaf tissue) were tested side to side. Results demonstrated APC‐RRV is a safe, cloneable and reliable approach subjected to quality control with application in quarantine surveillance and routine diagnostics of RRV.

Rickettsial DNA and a trans-splicing rRNA group I intron in the unorthodox mitogenome of the fern Haplopteris ensiformis

Plant mitochondrial genomes can be complex owing to highly recombinant structures, lack of gene syntenies, heavy RNA editing and invasion of chloroplast, nuclear or even foreign DNA by horizontal gene transfer (HGT). Leptosporangiate ferns remained the last major plant clade without an assembled mitogenome, likely owing to a demanding combination of the above. We here present both organelle genomes now for Haplopteris ensiformis. More than 1,400 events of C-to-U RNA editing and over 500 events of reverse U-to-C edits affect its organelle transcriptomes. The Haplopteris mtDNA is gene-rich, lacking only the ccm gene suite present in ancestral land plant mitogenomes, but is highly unorthodox, indicating extraordinary recombinogenic activity. Although eleven group II introns known in disrupted trans-splicing states in seed plants exist in conventional cis-arrangements, a particularly complex structure is found for the mitochondrial rrnL gene, which is split into two parts needing reassembly on RNA level by a trans-splicing group I intron. Aside from ca. 80 chloroplast DNA inserts that complicated the mitogenome assembly, the Haplopteris mtDNA features as an idiosyncrasy 30 variably degenerated protein coding regions from Rickettiales bacteria indicative of heavy bacterial HGT on top of tRNA genes of chlamydial origin.

Advances in Nucleotide Repeat Expansion Diseases: Transcription Gets in Phase.

Unstable DNA repeat expansions and insertions have been found to cause more than 50 neurodevelopmental, neurodegenerative, and neuromuscular disorders. One of the main hallmarks of repeat expansion diseases is the formation of abnormal RNA or protein aggregates in the neuronal cells of affected individuals. Recent evidence indicates that alterations of the dynamic or material properties of biomolecular condensates assembled by liquid/liquid phase separation are critical for the formation of these aggregates. This is a thermodynamically-driven and reversible local phenomenon that condenses macromolecules into liquid-like compartments responsible for compartmentalizing molecules required for vital cellular processes. Disease-associated repeat expansions modulate the phase separation properties of RNAs and proteins, interfering with the composition and/or the material properties of biomolecular condensates and resulting in the formation of abnormal aggregates. Since several repeat expansions have arisen in genes encoding crucial players in transcription, this raises the hypothesis that wide gene expression dysregulation is common to multiple repeat expansion diseases. This review will cover the impact of these mutations in the formation of aberrant aggregates and how they modify gene transcription.

Novel R2R3-MYB Transcription Factor LiMYB75 Enhances Leaf Callus Regeneration Efficiency in Lagerstroemia indica

Lagerstroemia indica is an important woody ornamental plant worldwide. However, the application of many technologies, such as transgenic breeding and genome editing, has been severely hampered due to the lack of efficient calli induction and regeneration technology. Here, we discussed a reliable and efficient calli induction and regeneration protocol using whole-leaf explants. This protocol’s effectiveness for the calli induction and regeneration systems in crape myrtle were up to 70.33% and 44.33%, respectively. Next, an efficient and stable Agrobacterium-mediated genetic transformation system was created from leaf calli, and the green fluorescent protein (GFP) was able to detect up to 90% of its positive frequency. Meanwhile, two positive lines’ transfer DNA insertion sites and directions were identified using whole genome sequencing. LiMYB75, a novel R2R3-MYB transcription factor, was identified and transferred to the L. indica genome to enhance the leaf calli regeneration frequency. Surprisingly, overexpressing LiMYB75 increased the frequency of calli regeneration in the leaf by 1.27 times and the number of regenerated plantlets per callus by 4.00 times compared to the wild type, by regulating the expression levels of genes involved in callus formation, such as SHOOT MERISTEMLESS (STM). Overall, our findings revealed a simple, reliable, and highly efficient transformation approach and identified the desirable candidate gene LiMYB75, which improves L. indica’s calli regeneration efficiency.

Improving Agrobacterium tumefaciens-Mediated Genetic Transformation for Gene Function Studies and Mutagenesis in Cucumber (Cucumis sativus L.).

In the post-genomics era, Agrobacterium tumefaciens-mediated genetic transformation is becoming an increasingly indispensable tool for characterization of gene functions and crop improvement in cucumber (Cucumis sativus L.). However, cucumber transformation efficiency is still low. In this study, we evaluated the effects of several key factors affecting the shoot-regeneration rate and overall transformation efficiency in cucumber including genotypes, the age and sources of explants, Agrobacterium strains, infection/co-cultivation conditions, and selective agents. We showed that in general, North China cucumbers exhibited higher shoot-regeneration rate than US pickling or slicing cucumbers. The subapical ground meristematic regions from cotyledons or the hypocotyl had a similar shoot-regeneration efficiency that was also affected by the age of the explants. Transformation with the Agrobacterium strain AGL1 yielded a higher frequency of positive transformants than with GV3101. The antibiotic kanamycin was effective in selection against non-transformants or chimeras. Optimization of various factors was exemplified with the development of transgenic plants overexpressing the LittleLeaf (LL) gene or RNAi of the APRR2 gene in three cucumber lines. The streamlined protocol was also tested in transgenic studies in three additional genes. The overall transformation efficiency defined by the number of verified transgenic plants out of the number of seeds across multiple experiments was 0.2-1.7%. Screening among T1 OE transgenic plants identified novel, inheritable mutants for leaf or fruit color or size/shape, suggesting T-DNA insertion as a potential source of mutagenesis. The Agrobacterium-mediated transformation protocol from this study could be used as the baseline for further improvements in cucumber transformation.

Assembly-free quantification of vagrant DNA inserts.

Inserts of DNA from extranuclear sources, such as organelles and microbes, are common in eukaryote nuclear genomes. However, sequence similarity between the nuclear and extranuclear DNA, and a history of multiple insertions, make the assembly of these regions challenging. Consequently, the number, sequence and location of these vagrant DNAs cannot be reliably inferred from the genome assemblies of most organisms. We introduce two statistical methods to estimate the abundance of nuclear inserts even in the absence of a nuclear genome assembly. The first (intercept method) only requires low-coverage (<1×) sequencing data, as commonly generated for population studies of organellar and ribosomal DNAs. The second method additionally requires that a subset of the individuals carry extranuclear DNA with diverged genotypes. We validated our intercept method using simulations and by re-estimating the frequency of human NUMTs (nuclear mitochondrial inserts). We then applied it to the grasshopper Podisma pedestris, exceptional for both its large genome size and reports of numerous NUMT inserts, estimating that NUMTs make up 0.056% of the nuclear genome, equivalent to >500 times the mitochondrial genome size. We also re-analysed a museomics data set of the parrot Psephotellus varius, obtaining an estimate of only 0.0043%, in line with reports from other species of bird. Our study demonstrates the utility of low-coverage high-throughput sequencing data for the quantification of nuclear vagrant DNAs. Beyond quantifying organellar inserts, these methods could also be used on endosymbiont-derived sequences. We provide an R implementation of our methods called "vagrantDNA" and code to simulate test data sets.

Comparison of truncated human angiotensin-converting enzyme 2 (hACE2) expression in pET28a(+) versus pET-SUMO vector and two Escherichia coli strains

PurposeTruncated human angiotensin-converting enzyme 2 (hACE2) expression rises a great scientific interest, considering its possible therapeutic and diagnostic applications. A promising research direction is the therapeutic use of smaller hACE2 versions with high binding affinity as decoy receptors for S1 glycoprotein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Another possible application is the use of these truncated versions for the functionalization of appropriate nanomaterials for constructing novel biosensors with a rapid and sensitive response for coronavirus disease 2019 (COVID-19) detection. The present study aimed to find a suitable system for high yield expression of different versions of truncated hACE2. Materials and methodsThe encoding DNA for the hACE2 fragments (7–507 aa, 16–128 aa, and 30–357 aa) was obtained by PCR amplification using as template pcDNA3.1-hACE2 plasmid and further cloned into pET28a(+) and pET-SUMO vectors. The positive clones were selected and the correct DNA insertion was confirmed through gene sequencing. The truncated hACE2 proteins were further expressed in two E. coli strains, Rosetta(DE3) and BL21(DE3). ResultsFor all three truncated hACE2 mini proteins, pET28a(+) does not lead to protein expression, regardless of the bacterial strain. The situation changes with the use of the pET-SUMO expression system when all hACE2 fragments are expressed, but with higher efficiency in E. coli BL21(DE3) than E. coli Rosetta. ConclusionIn the present study, we showed that different versions of recombinant hACE2 are successfully expressed in E. coli BL21(DE3) by using pET-SUMO expression system.

PASTE: The Way Forward for Large DNA Insertions.

PASTE: The Way Forward for Large DNA Insertions.

Application of DNA-free CRISPR/Cas-mediated Genome Editing in Crops: A Review

Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR-Cas9) system was instigated first into eukaryotes in the last ten years are becoming productive and worldwide application for genome modification. Genome engineering through insertion of foreign DNA insert have numerous disadvantages which could be overcome by use of DNA-free genome editing. Various ways of DNA-free genome editing mediated by CRISPR/Cas systems are CRISPR/Cas delivery as ribonucleoprotein, delivery of CRISPR/Cas as virus-like particles and agrobacterium-based delivery of CRISPR/Cas. Crop improvement through DNA-free genome editing via CRISPR/Cas have been applied in rice, wheat, maize, tomato, soybean and rare species like Nicotiana benthamiana etc. It is method of choice for precise genome editing without genome shuffle in an organism.

Versatile mapping-by-sequencing with Easymap v.2.

Mapping-by-sequencing combines Next Generation Sequencing (NGS) with classical genetic mapping by linkage analysis to establish gene-to-phenotype relationships. Although numerous tools have been developed to analyze NGS datasets, only a few are available for mapping-by-sequencing. One such tool is Easymap, a versatile, easy-to-use package that performs automated mapping of point mutations and large DNA insertions. Here, we describe Easymap v.2, which also maps small insertion/deletions (InDels), and includes workflows to perform QTL-seq and variant density mapping analyses. Each mapping workflow can accommodate different experimental designs, including outcrossing and backcrossing, F2, M2, and M3 mapping populations, chemically induced mutation and natural variant mapping, input files containing single-end or paired-end reads of genomic or complementary DNA sequences, and alternative control sample files in FASTQ and VCF formats. Easymap v.2 can also be used as a variant analyzer in the absence of a mapping algorithm and includes a multi-threading option.