RESUMEN
Insects pose significant challenges in cotton-producing regions. Here, they describe a high-throughput CRISPR/Cas9-mediated large-scale mutagenesis library targeting endogenous insect-resistance-related genes in cotton. This library targeted 502 previously identified genes using 968 sgRNAs, generated ≈2000 T0 plants and achieved 97.29% genome editing with efficient heredity, reaching upto 84.78%. Several potential resistance-related mutants (10% of 200 lines) their identified that may contribute to cotton-insect molecular interaction. Among these, they selected 139 and 144 lines showing decreased resistance to pest infestation and targeting major latex-like protein 423 (GhMLP423) for in-depth study. Overexpression of GhMLP423 enhanced insect resistance by activating the plant systemic acquired resistance (SAR) of salicylic acid (SA) and pathogenesis-related (PR) genes. This activation is induced by an elevation of cytosolic calcium [Ca2+ ]cyt flux eliciting reactive oxygen species (ROS), which their demoted in GhMLP423 knockout (CR) plants. Protein-protein interaction assays revealed that GhMLP423 interacted with a human epidermal growth factor receptor substrate15 (EPS15) protein at the cell membrane. Together, they regulated the systemically propagating waves of Ca2+ and ROS, which in turn induced SAR. Collectively, this large-scale mutagenesis library provides an efficient strategy for functional genomics research of polyploid plant species and serves as a solid platform for genetic engineering of insect resistance.
Asunto(s)
Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas , Humanos , Animales , Sistemas CRISPR-Cas/genética , Especies Reactivas de Oxígeno/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , InsectosRESUMEN
BACKGROUND: The exploitation of the CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) is becoming the method of choice for genome editing in E. coli. First proposed by Jiang and co-workers, the strategy has been subsequently fine-tuned by several authors who demonstrated, by using few selected loci, that the efficiency of mutagenesis (number of mutant colonies over total number of colonies analyzed) can be extremely high (up to 100%). However, from published data it is difficult to appreciate the robustness of the technology, defined as the number of successfully mutated loci over the total number of targeted loci. This information is particularly relevant in high-throughput genome editing, where repetition of experiments to rescue missing mutants would be impractical. This work describes a "brute force" validation activity, which culminated in the definition of a robust, simple and rapid protocol for single or multiple gene deletions. RESULTS: We first set up our own version of the CRISPR/Cas9 protocol and then we evaluated the mutagenesis efficiency by changing different parameters including sequence of guide RNAs, length and concentration of donor DNAs, and use of single stranded and double stranded donor DNAs. We then validated the optimized conditions targeting 78 "dispensable" genes. This work led to the definition of a protocol, featuring the use of double stranded synthetic donor DNAs, which guarantees mutagenesis efficiencies consistently higher than 10% and a robustness of 100%. The procedure can be applied also for simultaneous gene deletions. CONCLUSIONS: This work defines for the first time the robustness of a CRISPR/Cas9-based protocol based on a large sample size. Since the technical solutions here proposed can be applied to other similar procedures, the data could be of general interest for the scientific community working on bacterial genome editing and, in particular, for those involved in synthetic biology projects requiring high throughput procedures.
Asunto(s)
Sistemas CRISPR-Cas , Escherichia coli/genética , Edición Génica , Familia de Multigenes , Eliminación de Gen , Genoma Bacteriano , Recombinación Homóloga , Mutagénesis , ARN Guía de Kinetoplastida , Recombinasas/metabolismo , Biología Sintética/métodosRESUMEN
Inserting artificially-generated 'DNA islands' into a genome has shed new light on why some DNA sequences are methylated and others are not.
Asunto(s)
Cromatina/química , Islas de CpG/genética , AnimalesRESUMEN
The majority of mammalian promoters are CpG islands; regions of high CG density that require protection from DNA methylation to be functional. Importantly, how sequence architecture mediates this unmethylated state remains unclear. To address this question in a comprehensive manner, we developed a method to interrogate methylation states of hundreds of sequence variants inserted at the same genomic site in mouse embryonic stem cells. Using this assay, we were able to quantify the contribution of various sequence motifs towards the resulting DNA methylation state. Modeling of this comprehensive dataset revealed that CG density alone is a minor determinant of their unmethylated state. Instead, these data argue for a principal role for transcription factor binding sites, a prediction confirmed by testing synthetic mutant libraries. Taken together, these findings establish the hierarchy between the two cis-encoded mechanisms that define the DNA methylation state and thus the transcriptional competence of CpG islands.