CRISPR/dCas9-Based Systems: Mechanisms and Applications in Plant Sciences

RNA-guided genomic transcriptional regulation tools, namely clustered regularly interspaced short palindromic repeats interference (CRISPRi) and CRISPR-mediated gene activation (CRISPRa), are a powerful technology for gene functional studies. Deriving from the CRISPR/Cas9 system, both systems consist of a catalytically dead Cas9 (dCas9), a transcriptional effector and a single guide RNA (sgRNA). This type of dCas9 is incapable to cleave DNA but retains its ability to specifically bind to DNA. The binding of the dCas9/sgRNA complex to a target gene results in transcriptional interference. The CRISPR/dCas9 system has been explored as a tool for transcriptional modulation and genome imaging. Despite its potential applications and benefits, the challenges and limitations faced by the CRISPR/dCas9 system include the off-target effects, protospacer adjacent motif (PAM) sequence requirements, efficient delivery methods and the CRISPR/dCas9-interfered crops being labeled as genetically modified organisms in several countries. This review highlights the progression of CRISPR/dCas9 technology as well as its applications and potential challenges in crop improvement.

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CRISPR/dCas9-based Systems: Mechanisms and Applications in Plant Sciences

10.20944/preprints202102.0033.v1 ◽

2021 ◽

Author(s):

Chou Khai Soong Karlson ◽

Siti Nurfadhlina Mohd Noor ◽

Nadja Nolte ◽

Boon Chin Tan

Keyword(s):

Genetically Modified Organisms ◽

Crop Improvement ◽

Gene Activation ◽

Delivery Methods ◽

Guide Rna ◽

Efficient Delivery ◽

Potential Applications ◽

Transcriptional Modulation ◽

Plant Sciences ◽

Target Effects

RNA-guided genomic transcriptional regulation tools, namely Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) and CRISPR-mediated gene activation (CRISPRa), are a powerful technology for the field of functional genomics. Deriving from the CRISPR/Cas9 system, both systems comprise a catalytically dead Cas9 (dCas9) and a single guide RNA (sgRNA). This type of dCas9 is incapable of cleaving DNA but retains its ability to specifically bind to DNA. The binding of the dCas9/sgRNA complex to a target gene results in transcriptional interference. The CRISPR/dCas9 system has been explored as a tool for transcriptional modulation and genome imaging. Despite its potential applications and benefits, the challenges and limitations faced by the CRISPR/dCas9 system include the off-target effects, PAM sequence requirement, efficient delivery methods, and the CRISPR/dCas9-interfered crops being labeled as genetically modified organisms in several countries. This review highlights the progression of CRISPR/dCas9 technology as well as its applications and potential challenges in crop improvement.

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A potential clue for the application of prime editing in tomato, a dicot plant

10.1101/2021.03.15.435378 ◽

2021 ◽

Author(s):

Tien Van Vu ◽

Jihae Kim ◽

Swati Das ◽

Jae-Yean Kim

Keyword(s):

High Frequency ◽

Mammalian Cells ◽

Genome Engineering ◽

Crop Improvement ◽

Plant Genome ◽

Guide Rna ◽

Genome Modification ◽

Potential Applications ◽

Targeted Deep Sequencing ◽

Synthetic Sequence

Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a priming extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. However, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. However, our data obtained by targeted deep sequencing also revealed inefficient PE activity in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. These data could also help explain the recent successes of some prime editors in plants using improved expression systems. Our work provides an important clue for the application of the PE approach in crop improvement.

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CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration

Nucleic Acids Research ◽

10.1093/nar/gkz267 ◽

2019 ◽

Vol 47 (13) ◽

pp. e74-e74 ◽

Cited By ~ 13

Author(s):

Vu Anh Truong ◽

Mu-Nung Hsu ◽

Nuong Thi Kieu Nguyen ◽

Mei-Wei Lin ◽

Chih-Che Shen ◽

...

Keyword(s):

Stem Cells ◽

Bone Healing ◽

Gene Activation ◽

3D Culture ◽

Calvarial Bone ◽

Guide Rna ◽

Ppar Γ ◽

Efficient Delivery ◽

Plasmid Transfection ◽

Engineered Cartilage

Abstract Calvarial bone healing remains difficult but may be improved by stimulating chondrogenesis of implanted stem cells. To simultaneously promote chondrogenesis and repress adipogenesis of stem cells, we built a CRISPRai system that comprised inactive Cas9 (dCas9), two fusion proteins as activation/repression complexes and two single guide RNA (sgRNA) as scaffolds for recruiting activator (sgRNAa) or inhibitor (sgRNAi). By plasmid transfection and co-expression in CHO cells, we validated that dCas9 coordinated with sgRNAa to recruit the activator for mCherry activation and also orchestrated with sgRNAi to recruit the repressor for d2EGFP inhibition, without cross interference. After changing the sgRNA sequence to target endogenous Sox9/PPAR-γ, we packaged the entire CRISPRai system into an all-in-one baculovirus for efficient delivery into rat bone marrow-derived mesenchymal stem cells (rBMSC) and verified simultaneous Sox9 activation and PPAR-γ repression. The activation/inhibition effects were further enhanced/prolonged by using the Cre/loxP-based hybrid baculovirus. The CRISPRai system delivered by the hybrid baculovirus stimulated chondrogenesis and repressed adipogenesis of rBMSC in 2D culture and promoted the formation of engineered cartilage in 3D culture. Importantly, implantation of the rBMSC engineered by the CRISPRai improved calvarial bone healing. This study paves a new avenue to translate the CRISPRai technology to regenerative medicine.

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Disabling Cas9 by an anti-CRISPR DNA mimic

10.1101/129627 ◽

2017 ◽

Cited By ~ 5

Author(s):

Jiyung Shing ◽

Fuguo Jiang ◽

Jun-Jie Liu ◽

Nicholas L. Bray ◽

Benjamin J. Rauch ◽

...

Keyword(s):

Mammalian Cells ◽

Gene Editing ◽

Adaptive Immune System ◽

Binding Mode ◽

Human Cells ◽

Guide Rna ◽

Protospacer Adjacent Motif ◽

Cas9 Protein ◽

Potential Applications ◽

Natural Inhibitors

CRISPR-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known and the potential applications for Cas9 inhibitor proteins in mammalian cells has not fully been established. We show here that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-EM structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif (PAM). Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on pre-formed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.

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Exosome/Liposome-like Nanoparticles: New Carriers for CRISPR Genome Editing in Plants

International Journal of Molecular Sciences ◽

10.3390/ijms22147456 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7456

Author(s):

Mousa A. Alghuthaymi ◽

Aftab Ahmad ◽

Zulqurnain Khan ◽

Sultan Habibullah Khan ◽

Farah K. Ahmed ◽

...

Keyword(s):

Genome Editing ◽

Viral Vectors ◽

Polymeric Materials ◽

Inorganic Nanoparticles ◽

Plant Genome ◽

Delivery Methods ◽

Detailed Consideration ◽

Crispr System ◽

Efficient Delivery ◽

Low Cytotoxicity

Rapid developments in the field of plant genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems necessitate more detailed consideration of the delivery of the CRISPR system into plants. Successful and safe editing of plant genomes is partly based on efficient delivery of the CRISPR system. Along with the use of plasmids and viral vectors as cargo material for genome editing, non-viral vectors have also been considered for delivery purposes. These non-viral vectors can be made of a variety of materials, including inorganic nanoparticles, carbon nanotubes, liposomes, and protein- and peptide-based nanoparticles, as well as nanoscale polymeric materials. They have a decreased immune response, an advantage over viral vectors, and offer additional flexibility in their design, allowing them to be functionalized and targeted to specific sites in a biological system with low cytotoxicity. This review is dedicated to describing the delivery methods of CRISPR system into plants with emphasis on the use of non-viral vectors.

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Signal Transduction in Leaf Senescence: Progress and Perspective

Plants ◽

10.3390/plants8100405 ◽

2019 ◽

Vol 8 (10) ◽

pp. 405 ◽

Cited By ~ 3

Author(s):

Salman Ahmad ◽

Yongfeng Guo

Keyword(s):

Signal Transduction ◽

Leaf Senescence ◽

Recent Progress ◽

Crop Improvement ◽

Cellular Level ◽

Future Directions ◽

Nutrient Remobilization ◽

Potential Applications ◽

Leaf Tissues ◽

Inducing Factors

Leaf senescence is a degenerative process that is genetically controlled and involves nutrient remobilization prior to the death of leaf tissues. Age is a key developmental determinant of the process along with other senescence inducing factors. At the cellular level, different hormones, signaling molecules, and transcription factors contribute to the regulation of senescence. This review summarizes the recent progress in understanding the complexity of the senescence process with primary focuses on perception and transduction of senescence signals as well as downstream regulatory events. Future directions in this field and potential applications of related techniques in crop improvement will be discussed.

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Enhancement of target specificity of CRISPR–Cas12a by using a chimeric DNA–RNA guide

Nucleic Acids Research ◽

10.1093/nar/gkaa605 ◽

2020 ◽

Vol 48 (15) ◽

pp. 8601-8616 ◽

Cited By ~ 1

Author(s):

Hanseop Kim ◽

Wi-jae Lee ◽

Yeounsun Oh ◽

Seung-Hun Kang ◽

Junho K Hur ◽

...

Keyword(s):

Dna Sequences ◽

Genome Engineering ◽

Target Genes ◽

Target Sequence ◽

Energy Potential ◽

Guide Rna ◽

Sequence Recognition ◽

Protospacer Adjacent Motif ◽

Effective Manner ◽

Target Dna

Abstract The CRISPR–Cas9 system is widely used for target-specific genome engineering. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

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Functionalized lipid-like nanoparticles for in vivo mRNA delivery and base editing

Science Advances ◽

10.1126/sciadv.abc2315 ◽

2020 ◽

Vol 6 (34) ◽

pp. eabc2315 ◽

Cited By ~ 1

Author(s):

Xinfu Zhang ◽

Weiyu Zhao ◽

Giang N. Nguyen ◽

Chengxiang Zhang ◽

Chunxi Zeng ◽

...

Keyword(s):

Messenger Rna ◽

Low Dose ◽

Genetic Disorders ◽

Base Editing ◽

Guide Rna ◽

Efficient Delivery ◽

Human Factor Viii ◽

Mrna Therapeutics ◽

Further Development

Messenger RNA (mRNA) therapeutics have been explored to treat various genetic disorders. Lipid-derived nanomaterials are currently one of the most promising biomaterials that mediate effective mRNA delivery. However, efficiency and safety of this nanomaterial-based mRNA delivery remains a challenge for clinical applications. Here, we constructed a series of lipid-like nanomaterials (LLNs), named functionalized TT derivatives (FTT), for mRNA-based therapeutic applications in vivo. After screenings on the materials, we identified FTT5 as a lead material for efficient delivery of long mRNAs, such as human factor VIII (hFVIII) mRNA (~4.5 kb) for expression of hFVIII protein in hemophilia A mice. Moreover, FTT5 LLNs demonstrated high percentage of base editing on PCSK9 in vivo at a low dose of base editor mRNA (~5.5 kb) and single guide RNA. Consequently, FTT nanomaterials merit further development for mRNA-based therapy.

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Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of the catalytic domain of a hyperthermostable endo-1,4-β-D-mannanase fromThermotoga petrophilaRKU-1

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309110029131 ◽

2010 ◽

Vol 66 (9) ◽

pp. 1078-1081 ◽

Cited By ~ 6

Author(s):

Camila Ramos Santos ◽

Fabio Marcio Squina ◽

Andreia Meza Navarro ◽

Roberto Ruller ◽

Rolf Prade ◽

...

Keyword(s):

Catalytic Domain ◽

Unit Cell ◽

Unit Cell Parameters ◽

Structural Determinants ◽

Space Group ◽

X Ray ◽

Cell Parameters ◽

Functional Studies ◽

Thermotoga Petrophila ◽

Potential Applications

Endo-1,4-β-D-mannanases play key roles in seed germination and fruit ripening and have recently received much attention owing to their potential applications in the food, detergent and kraft pulp industries. In order to delineate their structural determinants for specificity and stability, X-ray crystallographic investigations combined with detailed functional studies are being performed. In this work, crystals of the catalytic domain of a hyperthermostable endo-1,4-β-D-mannanase fromThermotoga petrophilaRKU-1 were obtained from three different conditions, resulting in two crystalline forms. Crystals from conditions with phosphate or citrate salts as precipitant (CryP) belonged to space groupP212121, with unit-cell parametersa= 58.76,b= 87.99,c= 97.34 Å, while a crystal from a condition with ethanol as precipitant (CryE) belonged to space groupI212121, with unit-cell parametersa= 91.03,b= 89.97,c= 97.89 Å. CryP and CryE diffracted to resolutions of 1.40 and 1.45 Å, respectively.

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