High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system
Pengcheng WangJun ZhangLin SunYizan MaJiao XuSijia LiangJinwu DengJiafu TanZhang Qing-huaLili TuHenry DaniellShuangxia JinXianlong Zhang
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Abstract:
Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologues. To efficiently achieve site/gene-specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has exerted broad site-specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2) and an endogenous gene GhCLA1 were chosen as targets. The DsRed2-edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7-100%. The mutation genotype was checked for both genes with Sanger sequencing. Barcode-based high-throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1-edited T0 plants and it matched well with Sanger sequencing results. No off-target editing was detected at the potential off-target sites. These results prove that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing.Keywords:
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Genome engineering is a powerful tool for a wide range of applications in biomedical research and medicine. The development of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has revolutionized the field of gene editing, thus facilitating efficient genome editing through the creation of targeted double-strand breaks of almost any organism and cell type. In addition, CRISPR-Cas9 technology has been used successfully for many other purposes, including regulation of endogenous gene expression, epigenome editing, live-cell labelling of chromosomal loci, edition of single-stranded RNA and high-throughput gene screening. The implementation of the CRISPR-Cas9 system has increased the number of available technological alternatives for studying gene function, thus enabling generation of CRISPR-based disease models. Although many mechanistic questions remain to be answered and several challenges have yet to be addressed, the use of CRISPR-Cas9-based genome engineering technologies will increase our knowledge of disease processes and their treatment in the near future.
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The ongoing advent of genome editing with programmable nucleases, including zinc-finger nuclease (ZFN), TAL effector nuclease (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated RNA-guided endonuclease Cas9 (CRISPR/Cas9), have spurred the hematopoietic stem cell gene therapy (HSC-GT). In particular, CRISPR/Cas9-mediated gene editing revealed promising outcomes in several preclinical disease models including inherited and neoplastic hematological diseases. In this review, we focused on the utilization of the CRISPR/Cas9 system as a possible treatment option for hemoglobinopathies and hematological tumors. We summarize the recent advances with CRISPR/Cas9 and its therapeutic potential for genome editing in cells from hematopoietic origin. We also critically discussed the limitations inherent to the CRISPR/Cas9 and possible alternatives for the improvement of genome editing.
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Genome editing using CRISPR-associated technology is widely used to modify the genomes rapidly and efficiently on specific DNA double-strand breaks (DSBs) induced by Cas9 endonuclease. However, despite swift advance in Cas9 engineering, structural basis of Cas9-recognition and cleavage complex remains unclear. Proper assembly of this complex correlates to effective Cas9 activity, leading to high efficacy of genome editing events. Here, we develop a CRISPR/Cas9-RAD51 plasmid constitutively expressing RAD51, which can bind to singlestranded DNA for DSB repair. We show that the efficiency of CRISPR-mediated genome editing can be significantly improved by expressing RAD51, responsible for DSB repair via homologous recombination (HR), in both gene knock-out and knock-in processes. In cells with CRISPR/Cas9-RAD51 plasmid, expression of the target genes (cohesin SMC3 and GAPDH) was reduced by more than 1.9-fold compared to the CRISPR/Cas9 plasmid for knock-out of genes. Furthermore, CRISPR/Cas9-RAD51 enhanced the knock-in efficiency of DsRed donor DNA. Thus, the CRISPR/Cas9-RAD51 system is useful for applications requiring precise and efficient genome edits not accessible to HR-deficient cell genome editing and for developing CRISPR/Cas9-mediated knockout technology. [BMB Reports 2023; 56(2): 102-107].
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Abstract Targeted genome editing using engineered nucleases such as ZFNs and TALENs has been rapidly replaced by the CRISPR/Cas9 (clustered, regulatory interspaced, short palindromic/ CRISPR-associated nuclease) system. CRISPR/Cas9 technology represents a significant improvement enabling a new level of targeting, efficiency and simplicity. Gene editing mediated by CRISPR/Cas9 has been recently used not only in bacteria but in many eukaryotic cells and organisms, from yeasts to mammals. Other modifications of the CRISPR-Cas9 system have been used to introduce heterologous domains to regulate gene expressions or label specific loci in various cell types. The review focuses not only on native CRISPR/Cas systems which evolved in prokaryotes as an endogenous adaptive defense mechanism against foreign DNA attacks, but also on the CRISPR/Cas9 adoption as a powerful tool for site-specific gene modifications in fungi, plants and mammals.
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The rapid development of programmable nuclease-based genome editing technologies has enabled targeted gene disruption and correction both in vitro and in vivo This revolution opens up the possibility of precise genome editing at target genomic sites to modulate gene function in animals and plants. Among several programmable nucleases, the type II clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated nuclease 9 (Cas9) system has progressed remarkably in recent years, leading to its widespread use in research, medicine and biotechnology. In particular, CRISPR-Cas9 shows highly efficient gene editing activity for therapeutic purposes in systems ranging from patient stem cells to animal models. However, the development of therapeutic approaches and delivery methods remains a great challenge for biomedical applications. Herein, we review therapeutic applications that use the CRISPR-Cas9 system and discuss the possibilities and challenges ahead.
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Gene modifications in animal models have been greatly facilitated through the application of targeted genome editing tools. The prokaryotic CRISPR/Cas9 type II genome editing system has recently been applied in cell lines and vertebrates. However, we still have very limited information about the efficiency of mutagenesis, germline transmission rates and off-target effects in genomes of model organisms. We now demonstrate that CRISPR/Cas9 mutagenesis in zebrafish is highly efficient, reaching up to 86.0%, and is heritable. The efficiency of the CRISPR/Cas9 system further facilitated the targeted knock-in of a protein tag provided by a donor oligonucleotide with knock-in efficiencies of 3.5-15.6%. Mutation rates at potential off-target sites are only 1.1-2.5%, demonstrating the specificity of the CRISPR/Cas9 system. The ease and efficiency of the CRISPR/Cas9 system with limited off-target effects make it a powerful genome engineering tool for in vivo studies.
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