CRISPR–Cas systems and applications for crop bioengineering DOI Creative Commons
Mireia Uranga, Ana Montserrat Martín‐Hernández, Nico De Storme

и другие.

Frontiers in Bioengineering and Biotechnology, Год журнала: 2024, Номер 12

Опубликована: Окт. 16, 2024

CRISPR–Cas technologies contribute to enhancing our understanding of plant gene functions, and the precise breeding crop traits. Here, we review latest progress in genome editing, focusing on emerging systems, DNA-free delivery methods, advanced editing approaches. By illustrating applications for improving performance food quality, highlight potential genome-edited crops sustainable agriculture security.

Язык: Английский

CRISPR–Cas applications in agriculture and plant research DOI
Aytug Tuncel, Changtian Pan,

Joshua S. Clem

и другие.

Nature Reviews Molecular Cell Biology, Год журнала: 2025, Номер unknown

Опубликована: Март 7, 2025

Язык: Английский

Процитировано

1
Viral delivery of an RNA-guided genome editor for transgene-free plant germline editing DOI Creative Commons
Trevor Weiss,

Maris Kamalu,

Honglue Shi

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Июль 19, 2024

Abstract Genome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation. Plant viruses, which naturally infect and spread to most tissues, present a promising system for reagents. But viruses have limited cargo capacities, restricting their ability carry large CRISPR-Cas systems. Here, we engineered tobacco rattle virus (TRV) the compact RNA-guided TnpB enzyme ISYmu1 its guide RNA. This innovation allowed transgene-free Arabidopsis thaliana in single step, with edits inherited subsequent generation. By overcoming traditional reagent barriers, this approach offers novel platform genome editing, can greatly accelerate biotechnology basic research.

Язык: Английский

Процитировано

5
Engineering an optimized hypercompact CRISPR/Cas12j‐8 system for efficient genome editing in plants DOI Creative Commons
Shasha Bai,

Xingyu Cao,

Lizhe Hu

и другие.

Plant Biotechnology Journal, Год журнала: 2025, Номер unknown

Опубликована: Янв. 12, 2025

Summary The Cas12j‐8 nuclease, derived from the type V CRISPR system, is approximately half size of Cas9 and recognizes a 5′‐TTN‐3′ protospacer adjacent motif sequence, thus potentially having broad application in genome editing for crop improvement. However, its efficiency remains low plants. In this study, we rationally engineered both crRNA nuclease. markedly improved When combined, they exhibited robust activity soybean rice, enabling target sites that were previously uneditable. Notably, certain sequences, was comparable to SpCas9 when targeting identical it outperformed Cas12j‐2 variant, nCas12j‐2, across all tested targets. Additionally, developed cytosine base editors based on Cas12j‐8, demonstrating an average increase 5.36‐ 6.85‐fold base‐editing (C T) compared with unengineered system plants, no insertions or deletions (indels) observed. Collectively, these findings indicate hypercompact CRISPR/Cas12j‐8 serves as efficient tool mediated by nuclease cleavage

Язык: Английский

Процитировано

0
The genome awakens: transposon-mediated gene regulation DOI
Ileana Tossolini, Regina Mencia, A. Arce

и другие.

Trends in Plant Science, Год журнала: 2025, Номер unknown

Опубликована: Март 1, 2025

Язык: Английский

Процитировано

0
Targeted mutagenesis in Arabidopsis and medicinal plants using transposon‐associated TnpB DOI Open Access
Zongyou Lv, Wen‐Hua Chen, Shiyuan Fang

и другие.

Journal of Integrative Plant Biology, Год журнала: 2024, Номер 66(10), С. 2083 - 2086

Опубликована: Авг. 7, 2024

The programmable nuclease TnpB is significantly smaller than Cas9, can edit genes in medicinal plants, including Artemisia annua, Salvia miltiorrhiza, Scutellaria baicalensis, Isatis indigotica, and Codonopsis pilosula, has potential uses molecular breeding to enhance crop yield quality. Medicinal plants produce valuable compounds, but often at low concentrations. Genome editing could be used increase the production of secondary metabolites plants. clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system emerged as a simple, widely method for gene However, identification new endonucleases improve its efficiency by expanding range target sequences, decreasing off-target effects, and, proteins, facilitating delivery genome-editing tools. RNA-guided Cas9 IscB (encoded transposon IS200/IS605 family) may share common ancestor with (Kapitonov et al., 2015), which encoded small family. recently been an efficient tool genome engineering Escherichia coli animal cells (Altae-Tran 2021; Karvelis 2021). employ multiple guides concurrently modify (Wang 2024), analogous mechanisms observed IscB. applicability plant non-human DNA yet explored. Here, we successfully model Arabidopsis (Arabidopsis thaliana) several RNA-directed that guided reRNA (right end element RNA), 20-bp region matching (Karvelis We developed construct was driven U6 polymerase III promoter expressed under control Ubiquitin1 (UBQ1) (Figure 1A). In addition, incorporated eukaryotic nuclear localization signals both termini ensure nucleus. To visualize TnpB, fused yellow fluorescent (YFP) generate YFP-TnpB construct. Nicotiana benthamiana leaves clear S1), consistent (Nekrasov 2013). Editing using (A) right RNA (reRNA):TnpB protoplast transformation, Agrobacterium tumefaciens-mediated transient stable transformation planta. (B) Diagram CHLI2 site. (C) Agarose gel electrophoresis chain reaction products from sites within protoplasts. Genomic digested EcoRV (lanes 1–3); undigested genomic shown lane 4. (D) Alignment reads showing edits CHLI2. wild-type sequence top. Bases have altered edited are marked red. TAM (transposon-associated motif) located next (at 5′ end). Targeted CYP71AV1 (E), SmTⅡAS (F), SbGUS (G), IsYUC2 (H), SPSS2 (I) pilosula protoplasts, respectively. (J) shows indel-inducing activity four targets, each spanning 20 nucleotides, ABCG39, NAP10, GL3, well A. annua TLR1 (mean ± SD, n = 3 independent experiments). (K) Distribution indel profiles genes. blue line represents deletions, while red insertions. green target, pink indicates TAM. (L). (M). recognized reRNA:TnpB or highlighted gray. underlined, changes Next, constructed vector assess Arabidopsis. generated guide selecting following transposon-associated motif (TAM) "TTGAT" MAGNESIUM CHELATASE SUBUNIT I2 (CHLI2, At5g45930) 1B). facilitate successful edits, designed include restriction enzyme site, allowing detection through analysis determine mutagenesis extracted it EcoRV. amplified digestion (PCR) sequenced Sanger sequencing (Table S1). By employing this technique, effectively eliminated unedited selectively enriched underwent subsequent sequencing. Using control, fragment protoplasts expressing reRNA:TnpB, whereas neither nor 1C). These results indicate capable presence reRNA. Analysis PCR revealed mutations induced not only also adjacent regions S2). Deletions were detected. verify our other genes, targeted GL3 editing. As expected, indels (insertions deletions) base pair substitutions these S3). evaluate rates vivo, evaluated expression constructs leaves. Mutations predominantly occurred close proximity sequence, findings S4). provide evidence vivo. investigated whether result similar level transgenic via floral dip method. Approximately 50 T1 seedlings obtained. Sequencing many near (Figures 1D, S5), previous studies TnpB-mediated E. human mutation 14% (seven mutant out plants). all showed chimeric phenotype, suggesting CRISPR/Cas9, induces individual cells. Additionally, some deletions insertions flanking S3A, C). generation (T2), chimerism mutants displayed pale-green phenotype resembling lines S6) (Mao relatively length (20 bp) enabled non-specific binding, resulting effects those CRISPR/Cas9. occurrence Arabidopsis, searched sequences TAIR10 (https://www.arabidopsis.org/). This search yielded 13 S2), shared 15 17 bp identical sequence. Within identified no mutations. obtain homozygous generation, egg cell-specific (egg-cell pro) drive expression. cell pro:Cas9 approximately 17%; however, obtained ability affected factors 2015). failed detect pro:TnpB population, 13% 1L, M). species pilosula. CYP71AV1, SmTⅡAS, SbGUS, IsYUC2, S. I. C. respectively, transiently transformed Consistent 1E–I). miltiorrhiza 1F), species. overall editing, performed high-throughput annua. vector, subjected cleavage (Liu 2019). 30%, 2%, 15%, respectively 1J). Intriguingly, induction NAP10 higher previously reported 2021) 1K). 2% Moreover, highly outside regions, lower deletion efficiencies 1K, S7). suggest might associated structure. further analyze mutational gene-edited examined 50-bp had acquired sites, revealing prevalence compared site S7), prior Fewer detected more distant locations sites. Figure S9A, rate C-to-G TLR reached 33.07% test 1. notably falling below 3% S8, S9). Recent indicated significant rice (Karmakar 2024; Li efficiency. Overall, suitable Our demonstrate inducing (only 1,227 bp), makes amenable manipulation utilization Besides changes, delete fragments. (TTGAT) two bases longer protospacer (PAM) contains three specific PAM SpCas9 (NGG). since easy create generating substitutions. Additional optimization needed perfect tool. Nevertheless, use work funded National Key Research Development Program China (grant no. 2022YFC3501700), Natural Science Foundation (32070332), Shanghai Local Technology Fund Central Government (YDZX20203100002948). thank Tgene Biotech (Shanghai) Co., Ltd. providing us data. authors declare conflict interest. Z.L. W.C. conceived entire research plan; W.C., X.W., B.D, S.F. most work; J.X. provided technical assistance; Z.L., X.W. wrote manuscript; L.Z. helped organization All read approved contents paper. Supporting Information found online supporting information tab article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13758/suppinfo S1. Localization nuclei cytoplasm S2. S3. creates S4. S5. S6. S7. depicted. signifies denotes indicating S8. substitution targets nt illustrated S9. (A)and Supplementary Table 1 Primer list. TTGAT 2 Potential Please note: publisher responsible content functionality any supplied authors. Any queries (other missing content) should directed corresponding author article.

Язык: Английский

Процитировано

3
Unlocking crops’ genetic potential: Advances in genome and epigenome editing of regulatory regions DOI

Namra Ali,

Shubhangi Singh,

Rohini Garg

и другие.

Current Opinion in Plant Biology, Год журнала: 2024, Номер 83, С. 102669 - 102669

Опубликована: Ноя. 26, 2024

Язык: Английский

Процитировано

2
CRISPR–Cas systems and applications for crop bioengineering DOI Creative Commons
Mireia Uranga, Ana Montserrat Martín‐Hernández, Nico De Storme

и другие.

Frontiers in Bioengineering and Biotechnology, Год журнала: 2024, Номер 12

Опубликована: Окт. 16, 2024

CRISPR–Cas technologies contribute to enhancing our understanding of plant gene functions, and the precise breeding crop traits. Here, we review latest progress in genome editing, focusing on emerging systems, DNA-free delivery methods, advanced editing approaches. By illustrating applications for improving performance food quality, highlight potential genome-edited crops sustainable agriculture security.

Язык: Английский

Процитировано

0