9 research outputs found
CRISPR/Cas system: recent advances and prospects for genome editing
Genome editing (GE) has revolutionized biological research through the new ability to precisely edit the genomes of living organisms. In recent years, various GE tools have been explored for editing simple and complex genomes. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has widely been used in GE due to its high efficiency, ease of use, and accuracy. It can be used to add desirable and remove undesirable alleles simultaneously in a single event. Here, we discuss various applications of CRISPR/Cas9 in a range of important crops, compare it with other GE tools, and review its mechanism, limitations, and future possibilities. Various newly emerging CRISPR/Cas systems, including base editing (BE), xCas9, and Cas12a (Cpf1), are also considered
CRISPR/Cas systems in genome editing: methodologies and tools for sgRNA design, off-target evaluation and strategies to mitigate off-target effects
Life sciences have been revolutionized by genome editing (GE) tools, including zinc finger nucleases, transcription activatorâLike effector nucleases, and CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas (CRISPRâassociated) systems, which make the targeted modification of genomic DNA of all organisms possible. CRISPR/Cas systems are being widely used because of their accuracy, efficiency, and costâeffectiveness. Various classes of CRISPR/Cas systems have been developed, but their extensive use may be hindered by offâtarget effects. Efforts are being made to reduce the offâtarget effects of CRISPR/Cas9 by generating various CRISPR/Cas systems with high fidelity and accuracy. Several approaches have been applied to detect and evaluate the offâtarget effects. Here, the current GE tools, the offâtarget effects generated by GE technology, types of offâtarget effects, mechanisms of offâtarget effects, major concerns, and outcomes of offâtarget effects in plants and animals are summarized. The methods to detect offâtarget effects, tools for singleâguide RNA (sgRNA) design, evaluation and prediction of offâtarget effects, and strategies to increase the onâtarget efficiency and mitigate the offâtarget impact on intended genomeâediting outcomes are summarized
Disease Severity, Resistance Analysis, and Expression Profiling of Pathogenesis-Related Protein Genes after the Inoculation of Fusarium equiseti in Wheat
Wheat (Triticum aestivum) is an important cereal crop, grown throughout the temperate and in some tropical and sub-tropical zones, at higher elevations. Several biotic and abiotic factors influence the production of wheat. In the present study, two wheat varieties have been subjected to disease severity and resistance analysis against Fusarium equiseti. Disease severity analysis revealed Shafaq-2006 to be more resistant than Sahar-2006. Both varieties were further subjected to the expression analysis of six important defense-related genes by RT-PCR and quantitative real-time PCR. This analysis revealed that PR-1, TLP, Chitinase, and β-1,3-glucanase genes were highly expressed in Shafaq-2006 and possibly play a significant role in its defense mechanism. In addition, biochemical and physiochemical parameters were also studied to further explore the difference between resistant and susceptible varieties. With total proline and protein contents, sugar and chlorophyll contents also increased significantly in resistant variety. Likewise, higher relative water content, total plant length, and the high root–shoot ratio was observed in resistant plants, compared to susceptible wheat plants. These increases in chemical and physiological parameters might be related to the activation of the defense mechanism due to the higher expression of PR genes in the resistant wheat variety. These genes can further be employed for cloning into wheat and other transgenic crops to develop resistance against F. equiseti
Whole genome sequencing reveals rare off-target mutations and considerable inherent genetic and/or somaclonal variations in CRISPR/Cas9-edited cotton plants
The CRISPR/Cas9 system has been extensively applied for crop improvement. However, our understanding of Cas9 specificity is very limited in Cas9âedited plants. To identify onâ and offâtarget mutation in an edited crop, we described whole genome sequencing (WGS) of 14 Cas9âedited cotton plants targeted to three genes, and 3 negative (Ne) control and 3 wildâtype (WT) plants. In total, 4,188 â 6,404 unique single nucleotide polymorphisms (SNPs) and 312 â 745 insertions/deletions (indels) were detected in 14 Cas9âedited plants compared with WT, negative and cotton reference genome sequences. Since the majority of these variations lack a protospacerâadjacent motif (PAM), we demonstrated that the most variations following Cas9âedited are due either to somaclonal variation or/and preâexisting/natural variation from maternal plants, but not offâtarget effects. Of a total of 4,413 potential offâtarget sites (allowing ⤠5 mismatches within the 20âbp sgRNA and 3âbp PAM sequences), the WGS data revealed that only 4 are bona fide offâtarget indel mutations, validated by Sanger sequencing. Moreover, inherent genetic variation of WT can generate novel offâtarget sites and destroy PAMs, which suggested great care should be taken to design sgRNA for the minimizing of offâtarget effect. These findings suggested that CRISPR/Cas9 system is highly specific for cotton plants
High efficient and precise base editing of Câ˘G to Tâ˘A in the allotetraploid cotton (Gossypium hirsutum) genome using a modified CRISPR/Cas9 system
The baseâediting technique using CRISPR/nCas9 (Cas9 nickase) or dCas9 (deactivated Cas9) fused with cytidine deaminase is a powerful tool to create point mutations. In this study, a novel G. hirsutumâBase Editor 3 (GhBE3) base editing system has been developed to create single base mutations in the allotetraploid genome of cotton (Gossypium hirsutum). A cytidine deaminase sequence (APOBEC) fused with nCas9 and uracil glycosylase inhibitor (UGI) was inserted into our CRISPR/Cas9 plasmid (pRGEB32âGhU6.7). Three target sites were chosen for two target genes, GhCLA and GhPEBP, to test the efficiency and accuracy of GhBE3. The editing efficiency ranged from 26.67 to 57.78% at the three target sites. Targeted deep sequencing revealed that the CâT substitution efficiency within an âediting windowâ, approximately sixânucleotide windows of â17 to â12 bp from the PAM sequence, was up to 18.63% of the total sequences. The 27 most likely offâtarget sites predicted by CRISPRâP and CasâOFFinder tools were analyzed by targeted deep sequencing, and it was found that rare CâT substitutions (average <0.1%) were detected in the editing windows of these sites. Furthermore, wholeâgenome sequencing analyses on two GhCLAâedited and one wildâtype plants with about 100x depth showed that no bona fide offâtarget mutations were detectable from 1500 predicted potential offâtarget sites across the genome. In addition, the edited bases were inherited to T1 progeny. These results demonstrate that GhBE3 has high specificity and accuracy for the generation of targeted point mutations in allotetraploid cotton
Biological Applications of Ball-Milled Synthesized Biochar-Zinc Oxide Nanocomposite Using Zea mays L.
Nanotechnology is one of the vital and quickly developing areas and has several uses in various commercial zones. Among the various types of metal oxide-based nanoparticles, zinc oxide nanoparticles (ZnO NPs) are frequently used because of their effective properties. The ZnO nanocomposites are risk-free and biodegradable biopolymers, and they are widely being applied in the biomedical and therapeutics fields. In the current study, the biochar-zinc oxide (MB-ZnO) nanocomposites were prepared using a solvent-free ball-milling technique. The prepared MB-ZnO nanocomposites were characterized through scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, X-ray powder diffraction (XRD), and thermogravimetric analysis (TGA), Fourier-transform infrared spectroscopy (FTIR), and ultraviolet–visible (UV) spectroscopy. The MB-ZnO particles were measured as 43 nm via the X-ray line broadening technique by applying the Scherrer equation at the highest peak of 36.36°. The FTIR spectroscope results confirmed MB-ZnO’s formation. The band gap energy gap values of the MB-ZnO nanocomposites were calculated as 2.77 eV by using UV–Vis spectra. The MB-ZnO nanocomposites were tested in various in vitro biological assays, including biocompatibility assays against the macrophages and RBCs and the enzymes’ inhibition potential assay against the protein kinase, alpha-amylase, cytotoxicity assays of the leishmanial parasites, anti-inflammatory activity, antifungal activity, and antioxidant activities. The maximum TAC (30.09%), TRP (36.29%), and DPPH radicals’ scavenging potential (49.19%) were determined at the maximum dose of 200 µg/mL. Similarly, the maximum activity at the highest dose for the anti-inflammatory (76%), at 1000 μg/mL, alpha-amylase inhibition potential (45%), at 1000 μg/mL, antileishmanial activity (68%), at 100 μg/mL, and antifungal activity (73 ± 2.1%), at 19 mg/mL, was perceived, respectively. It did not cause any potential harm during the biocompatibility and cytotoxic assay and performed better during the anti-inflammatory and antioxidant assay. MB-ZnO caused moderate enzyme inhibition and was more effective against pathogenic fungus. The results of the current study indicated that MB-ZnO nanocomposites could be applied as effective catalysts in various processes. Moreover, this research provides valuable and the latest information to the readers and researchers working on biopolymers and nanocomposites
Novel and emerging biotechnological crop protection approaches
Traditional breeding or genetically modified organisms (GMOs) have for a long time been the sole approaches to effectively cope with biotic and abiotic stresses and implement the quality traits of crops. However, emerging diseases as well as unpredictable climate changes affecting agriculture over the entire globe force scientists to find alternative solutions required to quickly overcome seasonal crises. In this review, we first focus on cisgenesis and genome editing as challenging biotechnological approaches for breeding crops more tolerant to biotic and abiotic stresses. In addition, we take into consideration a toolbox of new techniques based on applications of RNA interference and epigenome modifications, which can be adopted for improving plant resilience. Recent advances in these biotechnological applications are mainly reported for nonâmodel plants and woody crops in particular. Indeed, the characterization of RNAi machinery in plants is fundamental to transform available information into biologically or biotechnologically applicable knowledge. Finally, here we discuss how these innovative and environmentally friendly techniques combined with traditional breeding can sustain a modern agriculture and be of potential contribution to climate change mitigation