<scp>CRISPR RNA</scp>‐guided integrase enables high‐efficiency targeted genome engineering in <i>Agrobacterium tumefaciens</i>

Aliu, Ephraim; Lee, Keunsub; Wang, Kan

doi:10.1111/pbi.13872

Cited by 17 publications

(20 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Plant genetic transformation is very important for basic biology research and modern plant breeding. At present, the Agrobacterium -mediated genetic transformation method is a widely used transformation technology [ 1 ]. Agrobacterium tumefaciens is a gram-negative soil bacterium that contains the Ti plasmid.…”

Section: Discussionmentioning

confidence: 99%

“…Plant genetic transformation is a key step in molecular design breeding, including genetic engineering and genome editing [ 1 ]. It can not only be used to identify gene functions but also to improve short plate traits of plant varieties, especially for genetic improvement of target traits of tree varieties with a long breeding generation cycle, which can significantly shorten the breeding cycle [ 2 ].…”

Section: Introductionmentioning

confidence: 99%

“…This method introduces foreign DNA fragments or gene editors carried by Ti plasmids, Ri plasmids, and other carriers into host cells through Agrobacterium infection to study related gene functions or realize plant breeding. It is the most widely used genetic transformation technology and method, promoting the research progress of plant molecular breeding [ 1 , 8 , 9 ].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Effect of a suitable treatment period on the genetic transformation efficiency of the plant leaf disc method

et al. 2023

View full text Add to dashboard Cite

Background Agrobacterium tumefaciens-mediated leaf disc genetic transformation is an important way to achieve transgenics or gene editing. Ensuring stable and efficient genetic transformation is still an important problem in modern biology. It is assumed that the difference in the development status of genetic transformation cells of receptor materials is the main reason for the difference and instability of genetic transformation efficiency; the stable and efficient genetic transformation rate can be obtained by defining the appropriate treatment period of the receptor material and applying genetic transformation in a timely manner. Results Based on these assumptions, we studied and established an efficient and stable Agrobacterium-mediated plant transformation system with hybrid poplar (Populus alba × Populus glandulosa, 84 K) leaves, stem segments and tobacco leaves as the research objects. There were differences in the development process of leaf bud primordial cells from different explants, and the genetic transformation efficiency was significantly related to the cell development stage of the in vitro cultured materials. Among them, the genetic transformation rate of poplar and tobacco leaves was the highest on the 3rd and 2nd day of culture, reaching 86.6% and 57.3%, respectively. The genetic transformation rate of poplar stem segments was the highest on the 4th day of culture, reaching 77.8%. The best treatment period was from the development of leaf bud primordial cells to the S phase of the cell cycle. The number of cells detected using flow cytometry and 5-ethynyl-2ʹ-deoxyuridine (EdU) staining, the expression of cell cycle-related protein CDKB1; 2, CDKD1; 1, CYCA3; 4, CYCD1; 1, CYCD3; 2, CYCD6; 1, and CYCH; 1 of explants, and morphological changes of explants can be used as indicators to determine the appropriate treatment period for genetic transformation. Conclusions Our study provides a new and universal set of methods and characteristics to identify the S phase of the cell cycle and apply genetic transformation treatments at the appropriate time. Our results are of great significance for improving the efficiency and stability of plant leaf disc genetic transformation.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Effect of a suitable treatment period on the genetic transformation efficiency of the plant leaf disc method

et al. 2023

View full text Add to dashboard Cite

show abstract

“…These modifications are either in CRISPR vector systems, explant selection, media composition, or the addition of some regulators [ 35 , 36 , 38 , 39 ]. Recently, to enhance the efficiency of Agrobacterium -mediated transformation for CRSIPR-Cas9, scientists have targeted the Agrobacterium genome [ 40 , 41 ] however, the technology faces multiple challenges that need to be improved further. Most recently some more advancements occurred in Agrobacterium -mediated transformations [ 26 , 42 , 43 ], in order to overcome these limitations, such as transgene-free editing and improvement in the delivery systems in plants [ 35 , 43 , 44 ].…”

Section: Ways To Deliver Crispr/cas Reagents To Plantsmentioning

confidence: 99%

Plant Virus-Derived Vectors for Plant Genome Engineering

Mahmood

Naqvi

Rahman

et al. 2023

Viruses

View full text Add to dashboard Cite

Advances in genome engineering (GE) tools based on sequence-specific programmable nucleases have revolutionized precise genome editing in plants. However, only the traditional approaches are used to deliver these GE reagents, which mostly rely on Agrobacterium-mediated transformation or particle bombardment. These techniques have been successfully used for the past decades for the genetic engineering of plants with some limitations relating to lengthy time-taking protocols and transgenes integration-related regulatory concerns. Nevertheless, in the era of climate change, we require certain faster protocols for developing climate-smart resilient crops through GE to deal with global food security. Therefore, some alternative approaches are needed to robustly deliver the GE reagents. In this case, the plant viral vectors could be an excellent option for the delivery of GE reagents because they are efficient, effective, and precise. Additionally, these are autonomously replicating and considered as natural specialists for transient delivery. In the present review, we have discussed the potential use of these plant viral vectors for the efficient delivery of GE reagents. We have further described the different plant viral vectors, such as DNA and RNA viruses, which have been used as efficient gene targeting systems in model plants, and in other important crops including potato, tomato, wheat, and rice. The achievements gained so far in the use of viral vectors as a carrier for GE reagent delivery are depicted along with the benefits and limitations of each viral vector. Moreover, recent advances have been explored in employing viral vectors for GE and adapting this technology for future research.

show abstract

“…The V. cholerae CAST, dubbed INTEGRATE, was later shown to enable site-specific transposon integration in Klebsiella oxytoca, Pseudomonas putida, and Agrobacterium fabrum. , For both CASTs, no host-specific genes are needed, potentially allowing for broader deployment than is typical for other CRISPR-enabled tools used in genome editing. In addition to the high efficiency of insertion, the ease with which the guide-RNA target can be changed to new targets makes this system extremely portable, enabling fast and efficient mutagenesis.…”

Section: Introductionmentioning

confidence: 99%

CRISPR-Associated Transposase for Targeted Mutagenesis in Diverse Proteobacteria

et al. 2023

View full text Add to dashboard Cite

Genome editing tools, through the disruption of an organism’s native genetic material or the introduction of non-native DNA, facilitate functional investigations to link genotypes to phenotypes. Transposons have been instrumental genetic tools in microbiology, enabling genome-wide, randomized disruption of genes and insertions of new genetic elements. Due to this randomness, identifying and isolating particular transposon mutants (i.e., those with modifications at a genetic locus of interest) can be laborious, often requiring one to sift through hundreds or thousands of mutants. Programmable, site-specific targeting of transposons became possible with recently described CRISPR-associated transposase (CASTs) systems, allowing the streamlined recovery of desired mutants in a single step. Like other CRISPR-derived systems, CASTs can be programmed by guide-RNA that is transcribed from short DNA sequence(s). Here, we describe a CAST system and demonstrate its function in bacteria from three classes of Proteobacteria. A dual plasmid strategy is demonstrated: (i) CAST genes are expressed from a broad-host-range replicative plasmid and (ii) guide-RNA and transposon are encoded on a high-copy, suicidal pUC plasmid. Using our CAST system, single-gene disruptions were performed with on-target efficiencies approaching 100% in Beta- and Gammaproteobacteria (Burkholderia thailandensis and Pseudomonas putida, respectively). We also report a peak efficiency of 45% in the Alphaproteobacterium Agrobacterium fabrum. In B. thailandensis, we performed simultaneous co-integration of transposons at two different target sites, demonstrating CAST’s utility in multilocus strategies. The CAST system is also capable of high-efficiency large transposon insertion totaling over 11 kbp in all three bacteria tested. Lastly, the dual plasmid system allowed for iterative transposon mutagenesis in all three bacteria without loss of efficiency. Given these iterative capabilities and large payload capacity, this system will be helpful for genome engineering experiments across several fields of research.

show abstract

CRISPR RNA‐guided integrase enables high‐efficiency targeted genome engineering in Agrobacterium tumefaciens

Cited by 17 publications

References 55 publications

Effect of a suitable treatment period on the genetic transformation efficiency of the plant leaf disc method

Effect of a suitable treatment period on the genetic transformation efficiency of the plant leaf disc method

Plant Virus-Derived Vectors for Plant Genome Engineering

CRISPR-Associated Transposase for Targeted Mutagenesis in Diverse Proteobacteria

Contact Info

Product

Resources

About