CRISPR/Cas9-based genome editing, with focus on transcription factors, for plant improvement

2020 ◽  
pp. 63-84
Author(s):  
Seema Pradhan ◽  
Chandra Kant ◽  
Vimal Pandey
Keyword(s):  
Transcription Factors ◽  
Genome Editing ◽  
Plant Improvement

scholarly journals In Vivo Study of Key Transcription Factors in Muscle Satellite Cells by CRISPR/Cas9/AAV9-sgRNA Mediated Genome Editing

2019 ◽  
Author(s):  
Liangqiang He ◽  
Yingzhe Ding ◽  
Yu Zhao ◽  
Karl K. So ◽  
Xianlu L. Peng ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Genome Editing ◽  
Satellite Cells ◽  
Muscle Regeneration ◽  
Molecular Mechanisms ◽  
Early Stage ◽  
Muscle Stem Cells ◽  
Muscle Satellite Cells ◽  
Regulatory Functions

ABSTRACTSkeletal muscle satellite cells (SCs) are adult muscle stem cells responsible for injury induced muscle regeneration. Despite advances in the knowledge of molecular mechanisms regulating SC lineage progression, our understanding of key transcription factors (TFs) and their regulatory functions in SCs in particularly the quiescent and early activation stages remains incomplete due to the lack of efficient method to screen and investigate the stage-specific key TFs. In this study, we succeeded in defining a distinct list of key TFs in early stages of SC fate transition using the paradigm of super enhancers (SEs). Particularly, leveraging the Cre-dependent Cas9 knockin mice and AAV9 mediated sgRNAs delivery, we generated a facile muscle specific genome editing system which allows gene depletion in SCs in vivo. Using MyoD locus as a proof of concept, we demonstrated that this CRISPR/Cas9/AAV9-sgRNA system can efficiently introduce mutagenesis at target locus and recapture the phenotypes reported in knockout mice. Further application of the system on key TFs, Myc, Bcl6 and Pknox2, revealed their distinct functions in the early stage of SC activation and damage induced muscle regeneration. Altogether our findings have proven the CRISPR/Cas9/AAV9-sgRNA system as a robust way for in vivo genome editing and elucidation of key factors governing SC activities.

scholarly journals Prospects for Knockout of MYB60, a Transcriptional Repressor of Anthocyanin Biosynthesis, in Brassicaceae Plants by Genome Editing

2022 ◽  
Author(s):  
Elena Mikhaylova ◽  
Alexander Artyukhin ◽  
Michael Shein ◽  
Khalit Musin ◽  
Anna Sukhareva ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Genome Editing ◽  
Agronomic Traits ◽  
Anthocyanin Biosynthesis ◽  
Gene Knockout ◽  
Transcriptional Repressor ◽  
Academic Community ◽  
Plant Responses ◽  
Individual Plant ◽  
Brassica Napus L

The Brassicaceae plant family contains many economically important crops such as Brassica napus L., Brassica rapa L., Brassica oleracea L., Brassica juncea L., Eruca sativa Mill., Camelina sativa L. and Raphanus sativus L. Insufficient data on the genetic regulation of agronomic traits in these species complicates the editing of their genomes. In recent years, the attention of the academic community has been drawn to anthocyanin hyperaccumulation. This trait is not only beneficial for human health, but can also increase plant resistance to stress. MYB transcription factors are the main regulators of flavonoid biosynthesis in plants. Some of them are well studied in Arabidopsis thaliana. The AtMYB60 gene is a transcriptional repressor of anthocyanin biosynthesis, and it also negatively impacts plant responses to drought stress. Myb60 is one of the least studied transcription factors with similar functions in Brassicaceae. There is a high degree of homology between predicted MYB60 genes of A. thaliana and related plant species. However, functions of these homologous genes have never been studied. Gene knockout by CRISPR/Cas technology remains the easiest way to perform genome editing in order to discover the role of individual plant genes. Disruption of genes acting as negative regulators of anthocyanin biosynthesis could result in color staining of plant tissues and an increase in stress tolerance. In the present study, we investigated the AtMYB60 gene and its homologs in Brassicaceae plants and suggested universal gRNAs to knockout these genes. Keywords: CRISPR, Brassicaceae, MYB60, knockout, anthocyanin

scholarly journals Krüppel-like factor gene function in the ctenophore Mnemiopsis leidyi assessed by CRISPR/Cas9-mediated genome editing

2019 ◽  
Author(s):  
Jason S Presnell ◽  
William E Browne
Keyword(s):  
Transcription Factors ◽  
Genome Editing ◽  
Gene Function ◽  
Functional Role ◽  
Cell Types ◽  
Mnemiopsis Leidyi ◽  
Morpholino Oligonucleotide ◽  
Animal Evolution ◽  
Animal Development ◽  
Summary Statement

AbstractThe Krüppel-like factor (Klf) gene family encodes for transcription factors that play an important role in the regulation of stem cell proliferation, cell differentiation, and development in bilaterians. While Klf genes have been shown to be expressed in various cell types in non-bilaterian animals, their functional role in early diverging animal lineages has not been assessed. Thus, the ancestral activity of these transcription factors in animal development is not well understood. The ctenophore Mnemiopsis leidyi has emerged as an important non-bilaterian model system for understanding early animal evolution. Here we characterize the expression and functional role of Klf genes during M. leidyi embryogenesis. Zygotic Klf gene function was assessed with both CRISPR/Cas9-mediated genome editing knockout and splice-blocking morpholino oligonucleotide knockdown approaches. Abrogation of zygotic Klf expression during M. leidyi embryogenesis results in the irregular development of several organs including the pharynx, tentacle bulbs, and apical organ. Our data suggest an ancient role for Klf genes in regulating endodermal patterning.Summary StatementUsing morpholino oligonucleotide knockdown and CRISPR/Cas9 genome editing, this study shows that in the ctenophore Mnemiopsis leidyi, tissues derived from the endoderm are dependent upon Klf5 ortholog expression for proper development and patterning.

scholarly journals Editing of the MYB genes in Brassica napus as a method to increase anthocyanin pigmentation and stress tolerance

2020 ◽  
Vol 224 ◽  
pp. 04022
Author(s):  
E V Mikhaylova ◽  
M. Y. Shein ◽  
A. Y. Artyukhin ◽  
A S Sukhareva ◽  
M. A. Panfilova ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Brassica Napus ◽  
Genome Editing ◽  
Anthocyanin Biosynthesis ◽  
Phytopathogenic Fungi ◽  
Anthocyanin Pigmentation ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Myb Genes ◽  
Genetic Constructs

Anthocyanin hyperaccumulation is an important agricultural trait, associated with resistance to abiotic stress, pests, phytopathogenic fungi and bacterial diseases. B. napus with increased anthocyanin pigmentation can be generated by genome editing. Many transcription factors of the MYB family are involved in stress response and anthocyanin biosynthesis. Genes AtMYB60, AtCPC and AtMYBL2 are negative regulators of anthocyanin biosynthesis in Arabidopsis, therefore the knockout of these genes can result in increased anthocyanin pigmentation. gRNA spacers were synthesized to target the orthologs of these genes, identified in Brassica napus. Resulting genetic constructs were introduced to the plant tissues by agroinfiltration. Transient expression of gRNAs targeting DNA-binding domains of MYB transcription factors along with Cas9 nuclease successfully promoted anthocyanin hyperaccumulation. These genetic constructs can be used for genome editing and production of new colored and stress tolerant varieties of oilseed rape.

Genome Editing Technologies for Plant Improvement: Advances, Applications and Challenges

2021 ◽  
pp. 213-240
Author(s):  
Rakesh Kumar ◽  
Narasimha Rao Nizampatnam ◽  
Minhaj Alam ◽  
Tarun K. Thakur ◽  
Anirudh Kumar
Keyword(s):  
Genome Editing ◽  
Plant Improvement

Modeling and targeting of erythroleukemia by hematopoietic genome editing

Blood ◽  
2021 ◽  
Author(s):  
Ilaria Iacobucci ◽  
Chunxu Qu ◽  
Elena Varotto ◽  
Laura J. Janke ◽  
Xu Yang ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Genome Editing ◽  
Clonal Evolution ◽  
Parp Inhibitor ◽  
Genomic Analysis ◽  
Genetic Alterations ◽  
Tumor Evolution ◽  
Preclinical Models ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Acute erythroid leukemia (AEL) is characterized by distinct morphology, mutational spectrum, a lack of preclinical models and poor prognosis. Here, using multiplexed genome editing of mouse hematopoietic stem and progenitor cells and transplant assay, we developed preclinical models of AEL and non-erythroid acute leukemia and demonstrated the central role of mutational cooperativity in determining leukemia lineage. Different combination of mutations in Trp53, Bcor, Dnmt3a, Rb1 and Nfix resulted in the development of leukemia with erythroid phenotype, and were accompanied by the acquisition of alterations in signaling and transcription factor genes that recapitulate human AEL by cross-species genomic analysis. Clonal expansion during tumor evolution was driven by mutational co-occurrence, with clones harboring a higher number of founder and secondary lesions (e.g. mutations in signaling genes) showing greater evolutionary fitness. Mouse and human AEL exhibited deregulation of genes regulating erythroid development, notably Gata1, Klf1, and Nfe2, driven by the interaction of mutations of the epigenetic modifiers Dnmt3a and Tet2 that perturbed methylation and thus expression of lineage-specific transcription factors. The established mouse leukemias were used as platform for drug screening. Drug sensitivity was associated with the leukemia genotype, with the PARP inhibitor talazoparib and the demethylating agent decitabine efficacious in Trp53/Bcor mutant AEL, CDK7/9 inhibitors in Trp53/Bcor/Dnmt3a mutant AEL and gemcitabine and bromodomain inhibitors in NUP98-KDM5A leukemia. In conclusion, combinatorial genome editing has demonstrated the interplay of founding and secondary genetic alterations in phenotype and clonal evolution, epigenetic regulation of lineage-specific transcription factors and therapeutic tractability in erythroid leukemogenesis.

scholarly journals Krüppel-like factor gene function in the ctenophore Mnemiopsis leidyi assessed by CRISPR/Cas9-mediated genome editing

Development ◽  
2021 ◽  
Author(s):  
Jason S. Presnell ◽  
William E. Browne
Keyword(s):  
Cell Proliferation ◽  
Transcription Factors ◽  
Genome Editing ◽  
Gene Function ◽  
Cell Types ◽  
Abnormal Development ◽  
Mnemiopsis Leidyi ◽  
Animal Evolution ◽  
Animal Development

The Krüppel-like factor (Klf) gene family encodes for transcription factors that play an important role in the regulation of stem cell proliferation, cell differentiation, and development in bilaterians. While Klf genes have been shown to functionally specify various cell types in non-bilaterian animals, their role in early diverging animal lineages has not been assessed. Thus, the ancestral activity of these transcription factors in animal development is not well understood. The ctenophore Mnemiopsis leidyi has emerged as an important non-bilaterian model system for understanding early animal evolution. Here we characterize the expression and functional role of Klf genes during M. leidyi embryogenesis. Zygotic Klf gene function was assessed with both CRISPR/Cas9-mediated genome editing and splice-blocking morpholino oligonucleotide knockdown approaches. Abrogation of zygotic Klf expression during M. leidyi embryogenesis results in abnormal development of several organs including the pharynx, tentacle bulbs, and apical organ. Our data suggest an ancient role for Klf genes in regulating endodermal patterning, possibly through regulation of cell proliferation.

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