Plant viral vectors: Expanding the Possibilities of Precise Gene Editing in Plant Genomes

Abstract The lack of a highly efficient method for delivering reagents for genome engineering to plant cells remains a bottleneck in achieving efficient gene-editing in plant genomes. A suite of recent reports uncovers the newly emerged roles of viral vectors, which can introduce gene-edits in plants with high mutation frequencies through in planta delivery. Here, we focus on the emerging protocols that utilized different approaches for virus-mediated genome editing in model plants. Testing of these protocols and the newly identified hypercompact Casɸ systems is needed to broaden the scope of genome-editing in most plant species, including crops, with minimized reliance on conventional plant transformation methods in the future.

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Advances in therapeutic application of CRISPR-Cas9

Briefings in Functional Genomics ◽

10.1093/bfgp/elz031 ◽

2019 ◽

Vol 19 (3) ◽

pp. 164-174 ◽

Cited By ~ 3

Author(s):

Jinyu Sun ◽

Jianchu Wang ◽

Donghui Zheng ◽

Xiaorong Hu

Keyword(s):

Gene Therapy ◽

Genome Editing ◽

Malignant Tumor ◽

Gene Editing ◽

Genome Engineering ◽

Therapeutic Application ◽

Adaptive Immune ◽

Efficient Gene

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is one of the most versatile and efficient gene editing technologies, which is derived from adaptive immune strategies for bacteria and archaea. With the remarkable development of programmable nuclease-based genome engineering these years, CRISPR-Cas9 system has developed quickly in recent 5 years and has been widely applied in countless areas, including genome editing, gene function investigation and gene therapy both in vitro and in vivo. In this paper, we briefly introduce the mechanisms of CRISPR-Cas9 tool in genome editing. More importantly, we review the recent therapeutic application of CRISPR-Cas9 in various diseases, including hematologic diseases, infectious diseases and malignant tumor. Finally, we discuss the current challenges and consider thoughtfully what advances are required in order to further develop the therapeutic application of CRISPR-Cas9 in the future.

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Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

Journal of Fungi ◽

10.3390/jof7070505 ◽

2021 ◽

Vol 7 (7) ◽

pp. 505

Author(s):

Ping Zhang ◽

Yu Wang ◽

Chenxi Li ◽

Xiaoyu Ma ◽

Lan Ma ◽

...

Keyword(s):

Genome Editing ◽

Fungal Pathogens ◽

Gene Editing ◽

Recombination Frequency ◽

Target Sequence ◽

Widespread Application ◽

Genetic Studies ◽

Efficient Gene ◽

Cryptococcus Species ◽

Overlap Pcr

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

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In planta particle bombardment (iPB): A new method for plant transformation and genome editing

Plant Biotechnology ◽

10.5511/plantbiotechnology.20.0206a ◽

2020 ◽

Vol 37 (2) ◽

pp. 171-176 ◽

Cited By ~ 1

Author(s):

Ryozo Imai ◽

Haruyasu Hamada ◽

Yuelin Liu ◽

Qianyan Linghu ◽

Yuya Kumagai ◽

...

Keyword(s):

Genome Editing ◽

Particle Bombardment ◽

Plant Transformation ◽

New Method ◽

In Planta

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Advances in Genome Editing With CRISPR Systems and Transformation Technologies for Plant DNA Manipulation

Frontiers in Plant Science ◽

10.3389/fpls.2020.637159 ◽

2021 ◽

Vol 11 ◽

Author(s):

Satya Swathi Nadakuduti ◽

Felix Enciso-Rodríguez

Keyword(s):

Genome Editing ◽

Viral Vectors ◽

Gene Editing ◽

De Novo ◽

Alternative Methods ◽

Zinc Finger Nucleases ◽

Target Range ◽

Homing Endonucleases ◽

Plant Gene ◽

Wide Range

The year 2020 marks a decade since the first gene-edited plants were generated using homing endonucleases and zinc finger nucleases. The advent of CRISPR/Cas9 for gene-editing in 2012 was a major science breakthrough that revolutionized both basic and applied research in various organisms including plants and consequently honored with “The Nobel Prize in Chemistry, 2020.” CRISPR technology is a rapidly evolving field and multiple CRISPR-Cas derived reagents collectively offer a wide range of applications for gene-editing and beyond. While most of these technological advances are successfully adopted in plants to advance functional genomics research and development of innovative crops, others await optimization. One of the biggest bottlenecks in plant gene-editing has been the delivery of gene-editing reagents, since genetic transformation methods are only established in a limited number of species. Recently, alternative methods of delivering CRISPR reagents to plants are being explored. This review mainly focuses on the most recent advances in plant gene-editing including (1) the current Cas effectors and Cas variants with a wide target range, reduced size and increased specificity along with tissue specific genome editing tool kit (2) cytosine, adenine, and glycosylase base editors that can precisely install all possible transition and transversion mutations in target sites (3) prime editing that can directly copy the desired edit into target DNA by search and replace method and (4) CRISPR delivery mechanisms for plant gene-editing that bypass tissue culture and regeneration procedures including de novo meristem induction, delivery using viral vectors and prospects of nanotechnology-based approaches.

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All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

10.1101/295055 ◽

2018 ◽

Cited By ~ 1

Author(s):

Raed Ibraheim ◽

Chun-Qing Song ◽

Aamir Mir ◽

Nadia Amrani ◽

Wen Xue ◽

...

Keyword(s):

Gene Therapy ◽

Neisseria Meningitidis ◽

Genome Editing ◽

Viral Vectors ◽

Genome Engineering ◽

Degradation Pathway ◽

Type I ◽

Guide Rna ◽

Hereditary Tyrosinemia

AbstractClustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: SpyCas9, SauCas9 and CjeCas9. However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors, especially those in which Cas9 and sgRNA are combined into a single vector genome. Here, we present an additional in vivo editing platform using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct PAM, making it an excellent candidate for safe gene therapy applications. We find that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogrammed the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we delivered NmeCas9 with its single-guide RNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded >35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo. Our findings indicate that NmeCas9 can facilitate future efforts to correct disease-causing mutations by expanding the targeting scope of RNA-guided nucleases.

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In planta genome editing with CRISPR/Cas9 ribonucleoproteins

10.1101/2021.06.24.449705 ◽

2021 ◽

Author(s):

Yuya Kumagai ◽

Yuelin Liu ◽

Haruyasu Hamada ◽

Weifeng Luo ◽

Jianghui Zhu ◽

...

Keyword(s):

Genome Editing ◽

Particle Bombardment ◽

Shoot Apical Meristem ◽

Apical Meristem ◽

Wheat Variety ◽

In Planta ◽

Culm Length ◽

Direct Delivery ◽

Transformation Methods

In planta genome editing represents an attractive approach to engineering crops/varieties that are recalcitrant to culture-based transformation methods. Here, we report the direct delivery of CRISPR/Cas9 ribonucleoproteins into the shoot apical meristem using in planta particle bombardment and introduction of a semidwarf1 (sd1)-orthologous mutation into wheat. The triple knockout tasd1 mutant of an elite wheat variety reduced culm length by 10% without a reduction in yield.

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Repurposing of Anthocyanin Biosynthesis for Plant Transformation and Genome Editing

Frontiers in Genome Editing ◽

10.3389/fgeed.2020.607982 ◽

2020 ◽

Vol 2 ◽

Author(s):

Yubing He ◽

Min Zhu ◽

Junhua Wu ◽

Lejun Ouyang ◽

Rongchen Wang ◽

...

Keyword(s):

Genome Editing ◽

Plant Species ◽

Plant Transformation ◽

Gene Editing ◽

Target Gene ◽

Target Genes ◽

Anthocyanin Biosynthesis ◽

Labor Time ◽

Visible Marker

CRISPR/Cas9 gene editing technology has been very effective in editing genes in many plant species including rice. Here we further improve the current CRISPR/Cas9 gene editing technology in both efficiency and time needed for isolation of transgene-free and target gene-edited plants. We coupled the CRISPR/Cas9 cassette with a unit that activates anthocyanin biosynthesis, providing a visible marker for detecting the presence of transgenes. The anthocyanin-marker assisted CRISPR (AAC) technology enables us to identify transgenic events even at calli stage, to select transformants with elevated Cas9 expression, and to identify transgene-free plants in the field. We used the AAC technology to edit LAZY1 and G1 and successfully generated many transgene-free and target gene-edited plants at T1 generation. The AAC technology greatly reduced the labor, time, and costs needed for editing target genes in rice.

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A simple method for spray-on gene editing in planta

10.1101/805036 ◽

2019 ◽

Cited By ~ 5

Author(s):

Cara Doyle ◽

Katie Higginbottom ◽

Thomas A. Swift ◽

Mark Winfield ◽

Christopher Bellas ◽

...

Keyword(s):

Carbon Dots ◽

Plant Transformation ◽

Gene Editing ◽

Production Systems ◽

Foliar Application ◽

Great Promise ◽

In Planta ◽

Crop Species ◽

Simple Method ◽

Negative Impacts

Potential innovation in Plant research using gene-edited and genetically modified plants is currently being hindered by inefficient and costly plant transformation. We show that carbon dots formed from natural materials (quasi-spherical, <10nm nanoparticles) can act as a fast vehicle for carrying plasmids into mature plant cells, resulting in transient plant transformation in a number of important crop species with no negative impacts on photosynthesis or growth. We further show that GFP, Cas9, and gRNA introduced into wheat via foliar application (spraying on) of plasmid coated carbon dots are expressed and, in the case of Cas9, make genome edits in SPO11 genes. Therefore, we present a protocol for spray-on gene editing that is simple, inexpensive, fast, transforms in planta, and is applicable to multiple crop species. We believe this technique creates many opportunities for the future of plant transformation in research and shows great promise for plant protein production systems.

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CRISPR/Cas12a mediated genome engineering in photosynthetic bacteria

10.1101/2020.10.05.327569 ◽

2020 ◽

Author(s):

Yang Zhang ◽

Jifeng Yuan

Keyword(s):

Genome Editing ◽

Transcriptional Repression ◽

Gene Editing ◽

Photosynthetic Bacteria ◽

Genome Engineering ◽

Biotechnological Applications ◽

End Joining ◽

Editing Efficiency ◽

Francisella Novicida ◽

Non Homologous End Joining

ABSTRACTPurple non-sulfur photosynthetic bacteria (PNSB) such as R. capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. In this study, we sought to develop the class II RNA-guided CRISPR/Cas12a system from Francisella novicida for both genome editing and gene down-regulation in R. capsulatus. About 90% editing efficiency was achieved by using CRISPR/Cas12a driven by a strong promoter Ppuc when targeting ccoO or nifH gene. When both genes were simultaneously targeted, the multiplex gene editing efficiency reached >63%. In addition, CRISPR interference using deactivated Cas12a was also evaluated using reporter genes gfp and lacZ, and the repression efficiency reached >80%. In summary, our work represents the first report to develop CRISPR/Cas12a mediated genome editing/transcriptional repression in R. capsulatus, which would greatly accelerate PNSB-related researches.IMPORTANCEPurple non-sulfur photosynthetic bacteria (PNSB) such as R. capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. However, lack of efficient gene editing tools remains a main obstacle for progressing in PNSB-related researches. Here, we developed CRISPR/Cas12a for genome editing via the non-homologous end joining (NHEJ) repair machinery in R. capsulatus. In addition, DNase-deactivated Cas12a was found to simultaneously suppress multiple targeted genes. Taken together, our work offers a new set of tools for efficient genome engineering in PNSB such as R. capsulatus.

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Plant-based biosensors for detecting CRISPR-mediated genome engineering

10.1101/2021.09.27.461852 ◽

2021 ◽

Author(s):

Guoliang Yuan ◽

Md Mahmudul Hassan ◽

Tao Yao ◽

Haiwei Lu ◽

Michael Melesse Vergara ◽

...

Keyword(s):

Gene Regulation ◽

Genome Editing ◽

Transient Expression ◽

Gene Editing ◽

Genome Engineering ◽

Protoplast Transformation ◽

Reliable System ◽

Base Editing ◽

Detection Systems ◽

Cas9 Nuclease

CRISPR/Cas has recently emerged as the most reliable system for genome engineering in various species. However, concerns about risks associated with CRISPR/Cas9 technology are increasing on potential unintended DNA changes that might accidentally arise from CRISPR gene editing. Developing a system that can detect and report the presence of active CRIPSR/Cas tools in biological systems is therefore very necessary. Here, we developed the real-time detection systems that can spontaneously indicate CRISPR-Cas tools for genome editing and gene regulation including CRISPR/Cas9 nuclease, base editing, prime editing and CRISPRa in plants. Using the fluorescence-based molecular biosensors, we demonstrated that the activities of CRISPR/Cas9 nuclease, base editing, prime editing and CRIPSRa can be effectively detected in transient expression via protoplast transformation and leaf infiltration (in Arabidopsis, poplar, and tobacco) and stable transformation in Arabidopsis.

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