Structural insights into distinct mechanisms of RNA polymerase II and III recruitment to snRNA promoters DOI Creative Commons
Syed Zawar Shah, Thomas N. Perry, Andrea Graziadei

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 10, 2024

Abstract RNA polymerase III (Pol III) is specialized in the transcription of short, essential RNAs, including U6 small nuclear RNAs (snRNAs). At snRNA genes, Pol recruited by Activating Protein Complex (SNAPc) forming, together with a Brf2-containing TFIIIB complex, transcriptionally competent pre-initiation complex (PIC). Additionally, SNAPc responsible for recruitment II at remaining snRNAs genes (U1, 2, 4 and 5), representing unique example multi subunit factor shared among different Polymerases. The mechanism cross-polymerase engagement role SNAPC2 SNAPC5 subunits remain poorly defined. Here, we present cryo-EM structures full-length SNAPc-containing PIC assembled on promoter open melting states 3.2-4.2Å resolution. Comparative structural analysis revealed unexpected differences yeast molecular basis selective structurally distinct within PICs. Harnessing crosslinking mass spectrometry, also localize proximity to bound DNA, expanding upon existing descriptions structure.

Language: Английский

Harnessing promoter elements to enhance gene editing in plants: perspectives and advances DOI Creative Commons
Nikita Gondalia, Luis Felipe Quiroz, Linyi Lai

et al.

Plant Biotechnology Journal, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 27, 2025

Summary Genome‐edited plants, endowed with climate‐smart traits, have been promoted as tools for strengthening resilience against climate change. Successful plant gene editing (GE) requires precise regulation of the GE machinery, a process controlled by promoters, which drives its transcription through interactions factors (TFs) and RNA polymerase. While constitutive promoters are extensively used in constructs, their limitations highlight need alternative approaches. This review emphasizes promise tissue/organ specific well inducible enable targeted spatiotemporal manner no effects on other tissues. Advances synthetic biology paved way creation offering refined control over expression augmenting potential GE. The integration these novel systems presents significant opportunities conditional genome editing. Moreover, advent bioinformatic artificial intelligence is revolutionizing characterization regulatory elements, enhancing our understanding roles plants. Thus, this provides insights into strategic use promoter to enhance precision, efficiency specificity GE, setting stage innovative crop improvement strategies.

Language: Английский

Citations

0
Optimizing the CRISPR/Cas9 system for gene editing in Yarrowia lipolytica DOI
Jianhui Liu,

Yamin Zhu,

Jin Hou

et al.

Engineering Microbiology, Journal Year: 2025, Volume and Issue: 5(2), P. 100193 - 100193

Published: March 18, 2025

Language: Английский

Citations

0
Computationally derived RNA polymerase III promoters enable maize genome editing DOI Creative Commons
É. Nagy, Ian Davis, Shanshan Song

et al.

Frontiers in Plant Science, Journal Year: 2025, Volume and Issue: 16

Published: March 19, 2025

CRISPR endonucleases require cognate non-coding RNA species for site-specific activity. These are typically expressed using endogenous polymerase III (Pol III) promoters compatible with the host species. This study describes applications of novel Pol promoters, which were computationally derived from a training set monocot U6 and U3 promoters. enabled genome editing in maize protoplast cells plants. Out 37 27 performed similarly to control promoter. Multiplexing five one construct simultaneous at unique sites single plant. Moreover, repeating same (crRNA) multiple improved up three-fold low-efficiency target site The ability derive on-demand increases flexibility efficiency maize.

Language: Английский

Citations

0
Efficient targeted T‐DNA integration for gene activation and male germline‐specific gene tagging in Arabidopsis DOI
Peng Xu, Jilei Huang, Xiaojing Chen

et al.

The Plant Journal, Journal Year: 2025, Volume and Issue: 121(6)

Published: March 1, 2025

SUMMARY Site‐specific DNA integration is an important tool in plant genetic engineering. Traditionally, this process relies on homologous recombination (HR), which known for its low efficiency cells. In contrast, Agrobacterium ‐mediated T‐DNA highly efficient transformation. However, typically inserted randomly into double‐strand breaks within the genome via non‐homologous end‐joining (NHEJ) repair pathway. study, we developed approach of CRISPR/Cas9‐mediated targeted Arabidopsis, was more rapid and than HR‐mediated method. This aided gene activation male germline‐specific tagging. Gene accomplished by positioning CaMV35S promoter at left border T‐DNA, thereby activating specific downstream genes. The FT MYB26 significantly increased their transcriptional expression, resulted early flowering altered pattern cell wall thickening anther endothelium, respectively. Male tagging incorporates two reporters, namely, NeoR MGH3::mCherry , T‐DNA. design facilitates creation insertional mutants, simplifies analysis mutated alleles, allows cellular tracking germline cells during fertilization. We successfully applied system to target GEX2. conclusion, our results demonstrated that site‐specific fragments can be rapidly efficiently achieved through NHEJ pathway, making broadly applicable various contexts.

Language: Английский

Citations

0
Optimization of CRISPR-Cas9 system in Eustoma grandiflorum DOI Creative Commons
Xueqi Li,

Fanqi Bu,

Lishan Wang

et al.

iScience, Journal Year: 2024, Volume and Issue: 27(3), P. 109053 - 109053

Published: Feb. 1, 2024

The optimization of the CRISPR-Cas9 system for enhancing editing efficiency holds significant value in scientific research. In this study, we optimized single guide RNA and

Language: Английский

Citations

3
Recalcitrance to transformation, a hindrance for genome editing of legumes DOI Creative Commons

V. M. Nivya,

Jasmine M. Shah

Frontiers in Genome Editing, Journal Year: 2023, Volume and Issue: 5

Published: Sept. 21, 2023

Plant genome editing, a recently discovered method for targeted mutagenesis, has emerged as promising tool crop improvement and gene function research. Many genome-edited plants, such rice, wheat, tomato, have over the last decade. As preliminary steps in procedure editing involve genetic transformation, amenability to depends on efficiency of engineering. Hence, there are numerous reports aforementioned crops because they transformed with relative ease. Legume rich protein and, thus, favored source plant proteins human diet most countries. However, legume cultivation often succumbs various biotic/abiotic threats, thereby leading high yield loss. Furthermore, certain legumes like peanuts possess allergens, these need be eliminated deprive many people from gaining benefits crops. Further variations limited legumes. Genome potential offer solutions not only combat stress but also generate desirable knock-outs variants. excluding soybean, alfalfa,

Language: Английский

Citations

7
Efficient Gene Editing and Overexpression of Gametophyte Transformation in a Model Fern DOI Creative Commons
Wei Jiang, Fenglin Deng, Mohammad Babla

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 11, 2024

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-related nuclease (Cas) system allows precise and easy editing of genes in many plant species. However, this has not yet been applied to any fern species due the complex characteristics genomes, genetics physiology. Here, we established, for first time, a protocol gametophyte-based screening single-guide RNAs (sgRNAs) with high efficiency CRISPR/Cas-mediated gene model species, Ceratopteris richardii . We utilized C. Actin promoter drive sgRNA expression enhanced CaMV 35S Streptococcus pyogenes Cas9 CRISPR-mediated system, which was employed successfully edit few (e.g., nucleotidase/phosphatase 1, CrSAL1 ; Cryptochrome 4, CRY4 ) CrPDS , encoding phytoene desaturase protein that resulted an albino phenotype Knockout significantly reduced stomatal conductance ( g s ), leaf transpiration rate E stomatal/pore length, abscisic acid (ABA)-induced reactive oxygen (ROS) accumulation guard cells. Moreover, overexpressing plants showed increased net photosynthetic A intrinsic water use iWUE as well most traits ROS production cells compared those wild-type (WT) plants. Taken together, optimized CRISPR/Cas9 provides useful tool functional genomics allowing exploration functions evolutionary biology, herbal medicine discovery agricultural applications.

Language: Английский

Citations

2
Towards DNA-free CRISPR/Cas9 genome editing for sustainable oil palm improvement DOI
Mat Yunus Abdul Masani,

Jamaludin Norfaezah,

Bohari Bahariah

et al.

3 Biotech, Journal Year: 2024, Volume and Issue: 14(6)

Published: May 28, 2024

Language: Английский

Citations

1
Investigating cold tolerance mechanisms in rice seedlings: Alternative splicing, promoter analysis, and their applications for marker development DOI Creative Commons

Patcharaporn Summat,

Keasinee Tongmark,

Sriprapai Chakhonkaen

et al.

Plant Stress, Journal Year: 2024, Volume and Issue: 13, P. 100530 - 100530

Published: July 8, 2024

Cold stress harms rice seedlings, causing yield reduction. Enhancing cold tolerance at the seedling stage is crucial for breeding. OsFH10, OsFER1, ONAC045, and OsProT were selected to study gene expression including alternative splicing promoter sequence analysis in seedlings. Under stress, four genes exhibited showing intron retention isoforms, displayed a consistent pattern of upregulation group cold-tolerant varieties, while no isoforms presented sensitive varieties. These might play role facilitating improved adaptation Promoter revealed polymorphisms Single nucleotide (SNPs) or Insertions Deletions (InDels) ONAC045 that distinguished from cold-sensitive SNP InDel markers developed validated using 159 lines. associated with (P < 0.05) practical use general agarose gel. The marker showed high efficacy predicting germplasm achieving an accuracy 43.8%. Furthermore, greater sensitivity towards indica Both have wide distribution all subspecies. Our findings elucidate response mechanisms provide insights into potential application developing

Language: Английский

Citations

1
Development and application of gene editing in citrus: how and what to do DOI Creative Commons
Shuai Wang,

Sailan Zhao,

Meixia Du

et al.

Horticulture Advances, Journal Year: 2024, Volume and Issue: 2(1)

Published: Dec. 10, 2024

Abstract Conventional breeding techniques have been effectively utilized for the enhancement of citrus varieties. Nonetheless, traits such as an extended juvenile phase, cross- or self-incompatibility, high genetic heterozygosity, and polyembryony posed significant challenges limitations to these methods. The clustered regularly interspaced short palindromic repeats (CRISPR) genome editing has progressively emerged a vital tool research. This article reviews array CRISPR/Cas systems, emphasizes recent advancements in using CRISPR/Cas, explores application this technology bolster resistance canker. review also covers development CRISPR/Cas-mediated transformation regeneration systems citrus, alongside approaches generating transgene-free germplasm. Moreover, regulatory landscape societal acceptance are examined. Lastly, potential applications proposed, with attention prospective challenges.

Language: Английский

Citations

1