Planta, Journal Year: 2023, Volume and Issue: 257(4)
Published: March 13, 2023
Language: Английский
Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)
Published: Sept. 29, 2023
Abstract Among CRISPR-Cas genome editing systems, Streptococcus pyogenes Cas9 (SpCas9), sourced from a human pathogen, is the most widely used. Here, through in silico data mining, we have established an efficient plant engineering system using CRISPR-Cas9 probiotic Lactobacillus rhamnosus . We confirmed predicted 5’-NGAAA-3’ PAM via bacterial depletion assay and showcased its exceptional efficiency rice, wheat, tomato, Larix cells, surpassing LbCas12a, SpCas9-NG, SpRY when targeting identical sequences. In stable rice lines, LrCas9 facilitates multiplexed gene knockout coding sequence achieves knockdown targeted promoter deletion, demonstrating high specificity. also developed LrCas9-derived cytosine adenine base editors, expanding capabilities. Finally, by harnessing LrCas9’s A/T-rich preference, created CRISPR interference activation systems plants. Together, our work establishes CRISPR-LrCas9 as user-friendly tool for diverse applications crops beyond.
Language: Английский
Citations
18
Journal of Integrative Plant Biology, Journal Year: 2024, Volume and Issue: 66(4), P. 638 - 641
Published: Feb. 13, 2024
The recent advancements in developing the CRISPR/Cas9 system and various derivative tools (e.g., base editors) have accelerated basic plant science research crop improvement by creating multiple types of genetic variations (Li et al., 2023a). However, use Cas9 protein is frequently limited requirement G/C-rich protospacer-adjacent motif (PAM) sequences, especially triticeae plants, many which are important food forage crops carrying large complex genomes. CRISPR/CasΦ (CRISPR/Cas12j) has recently been discovered from bacteriophages, prefers 5′-TBN-3′ PAMs suitable for specific biological therapeutic applications (Pausch 2020). With a smaller size (700–800 aa) than Cas12a (1,100 SpCas9 (1,300 aa), it valuable DNA editing where or nucleic acid limiting factor (Zhan 2021). Lately, CRISPR/CasΦ2 demonstrated useful gene transient transgenic experiments Arabidopsis, tomato, rice, maize (Liu 2022; Li 2023b). remains unclear whether may function plants. More importantly, worth exploring if this hypercompact be adopted precise plants (Figure 1A). Improving (A) CRISPR/CasΦ2-meadited toolkits. (B) Comparison frequency among pBlunt-CasΦ2Ta-V1/V2/V3/V4 at TaGW2 target sites (TBN PAMs) with TaU3-tRNA-crRNA wheat protoplasts. Representative deletion locations were summarized using TaGW2-crRNA-TTN/pBlunt-CasΦ2Ta-V3 data. (C) Assessment frequencies CRISPR/CasΦ2Ta-V3 CRISPR/VCasΦ2Ta-V3 TaGW2-crRNA-TTN site (D) Test TaGW2/ScPhyA-crRNA-TTN paired opposite crRNAs rye (E) Diagram CasΦ2/dCasΦ2 VCasΦ2/dVCasΦ2-based CBEs. (F) Frequencies C-to-T indels obtained four CBEs (G) frequencies, window five different pBlunt-dCasΦ2Ta-CBE (H) dCasΦ2-based adenine editor (ABE). (I) A-to-G inducing ABE (J) CasΦ2/dCasΦ2-derived cytosine editors (CBEs) TaGW2/TaPIN-crRNA-TTN dCasΦ2-derived TaALS-crRNA-TTN All values mean ± s.e.m. *P < 0.05, **P 0.01; ns, no significant difference two-tailed Student's t test. To address above questions, we first synthesized codon-optimized CasΦ2 constructed UBQ::CasΦ2Ta TaU3::crRNA cassette placed into pBlunt vector S1A). We chose genes editing: two (TaGW2 TaPIN) (ScPhyA ScPhyB). As B G, T C, designed three each TTN, TGN, TCN PAM (creating 12 sites), covering similar crRNA binding sites. When testing protoplasts-based systems, obvious was detected PCR/RE assays any S1B). This line very low efficiency (<1%) genome Arabidopsis Consequently, set out to improve CRISPR/CasΦ2. First, changed way processing tested nuclear localization signals (NLSs) CasΦ2. By combining TaU3 promoter-driven polycistronic-tRNA-crRNA NLSs (SV40 nucleoplasmin long NLS) fused CasΦ2Ta, generated versions CRISPR/CasΦ2Ta 1B, V2, V3, V4). protoplasts sites, found that pBlunt-TaU3-tRNA-crRNA/pBlunt-CasΦ2Ta-V3, one N- C-termini respectively, could most efficiently near-background level up 3.2% 1B). Considering CasΦ variants NCasΦ VCasΦ cleave substrate faster 2021), next prepared new constructs expressing NCasΦ2Ta-V3 VCasΦ2Ta-V3 their efficiencies ScPhyA protoplasts, appropriate pBlunt-TaU3-tRNA-crRNA S2A). showed both improved compared CasΦTa-V3, exhibiting an overall higher (2.5–6.0-fold increase, Figure S2B, C). Hence T-DNA constructs, pLH-CasΦ2Ta-V3 pLH-VCasΦ2Ta-V3, TaU3::tRNA-crRNA design S3A). Sanger sequencing analysis indicated pLH-VCasΦ2Ta-V3 induced 30% (6/20) T0 did (12.5%, 2/16) 1C), mutations being 3–27 bp deletions S3B). Analysis T1 verified inheritance indel pLH-VCasΦ2Ta-V3-TaGW2-crRNA-TTN generation (Table S3). Interestingly, observed crRNAs, arranged as 5′-TTN-N18-spacing-N18-YAA-3′, further pBlunt-VCasΦ2Ta-V3 assays. ScPhyA-crRNA-TTN, ~30 ~60-bp spacing sequences 1D). (~30 sequence) ~1.5-fold produced single Altogether, results suggest enables rye, potential crRNAs. Furthermore, endeavored develop (CBE ABE) CasΦ2Ta-V3. created catalytically inactive dCasΦ2Ta-V3 mutating active RuvC domain (D394, E606, D695) Then human APOBEC3A CasΦ2Ta-V3 (Zong 2018), pBlunt-CasΦ2Ta-CBE 1E). investigate VCasΦ2Ta influence CBE, also pBlunt-VCasΦ2Ta-CBE pBlunt-dVCasΦ2Ta-CBE vectors, dVCasΦ2Ta-V3 developed similarly dCasΦ2Ta-V3. site. Deep (up ~4%) levels 1F). expected, exhibited high Thus, another employed test In these assays, CBE ranged 1.9% 5.5%, spanning C2 C17 protospacers 1G), wider reported Cas9-based 2018). Therefore, pLH-dCasΦ2Ta-CBE pLH-CasΦ2Ta-CBE S5A), TaPIN-crRNA-TTN pLH-dCasΦ2Ta-CBE, activities 9.1% 6.9% TaPIN-crRNA-TTN, only C substitution 1J). contrast, mutants pLH-CasΦ2Ta-CBE-TaGW2-crRNA-TTN, 80% mostly transmitted For evaluating usefulness dCasΦ2-ABE, TadA8e XTEN linker (Yan thus generating pBlunt-dCasΦ2Ta-ABE 1H). investigated all targets (in TaALS, TaNAC2, ScPhyB, ScPhyC, respectively), ranging 0.8% 3.0% 1I). deamination spanned protospacer positions A9–A11, unwanted Hence, pLH-dCasΦ2Ta-ABE S6A) examine activity 6% (Figures 1J, S6B), Collectively, illustrated feasibility rye. Finally, examined off-targeting CRISPR/VCasΦ2Ta-V3, CRISPR/dCasΦ2Ta-CBE CRISPR/dCasΦ2Ta-ABE mediated mutants. used analysis, eight (1–4 mismatches; Table S4) identified common genome. revealed off-target events S7A, B), indicating specificity wheat. summary, changes expression, NLS incorporation, variants, resulting successful knockout specificity. proved time CRISPR/dCasΦ2-CBE CRISPR/dCasΦ2-ABE functional its unique properties, i.e., efficient TTN alternative window, provides complementary engineering tool, find wide future on CRISPR/CasΦ2-mediated modifications. work supported National Key Research Development Program China (2021YFF1000203) Natural Science Foundation (32000286 32370432). authors declare conflict interest. S.Z., X.H., Y.Z., Y.H., H.Liu., Z.C., H.Li., Dan.W., C.T., Y.Y., Y.G. performed experiments. X.J., Dao.W., X.S. conceived project wrote manuscript. approved final Additional Supporting Information online supporting information tab article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13624/suppinfo S1. pBlunt-TaU3-crRNA pBlunt-CasΦ2Ta-V1 vectors S2. pBlunt-CasΦ2Ta-V3, pBlunt-NCasΦ2Ta-V3 assessment S3. representative genotypes S4. strategy increasing pBlunt-VCasΦ2Ta-V3-mediated assay S5. diagram S6. S7. CRISPR/VCasΦ2Ta-V3-TaGW2-crRNA-TTN, CRISPR/dCasΦ2Ta-CBE-TaGW2-crRNA-TTN CRISPR/dCasΦ2Ta-ABE-TaALS-crRNA-TTN indel, primer constructing study primers preparing mutation transmission derived pLH-CasΦ2Ta-CBE, progenies Potential barcodes deep Other Please note: publisher not responsible content functionality supplied authors. Any queries (other missing content) should directed corresponding author article.
Language: Английский
Citations
6
International Journal of Molecular Sciences, Journal Year: 2024, Volume and Issue: 25(5), P. 2606 - 2606
Published: Feb. 23, 2024
The use of gene-editing tools, such as zinc finger nucleases, TALEN, and CRISPR/Cas, allows for the modification physiological, morphological, other characteristics in a wide range crops to mitigate negative effects stress caused by anthropogenic climate change or biotic stresses. Importantly, these tools have potential improve crop resilience increase yields response challenging environmental conditions. This review provides an overview techniques used plants, focusing on cultivated tomatoes. Several dozen genes that been successfully edited with CRISPR/Cas system were selected inclusion illustrate possibilities this technology improving fruit yield quality, tolerance pathogens, responses drought soil salinity, among factors. Examples are also given how domestication wild species can be accelerated using generate new better adapted climatic situation suited indoor agriculture.
Language: Английский
Citations
6
Plant Communications, Journal Year: 2024, Volume and Issue: 5(11), P. 101068 - 101068
Published: Aug. 23, 2024
Language: Английский
Citations
5
Plant Communications, Journal Year: 2024, Volume and Issue: 5(7), P. 100881 - 100881
Published: March 16, 2024
Language: Английский
Citations
4
Plant Communications, Journal Year: 2024, Volume and Issue: 5(6), P. 100921 - 100921
Published: April 15, 2024
CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement. However, the challenge of low activity complicates identification events. In this study, we introduce multiple single transcript unit surrogate reporter (STU-SR) systems to enhance selection genome-edited plants. These use same guide RNAs designed endogenous genes edit genes, establishing a direct link between gene that genes. Various strategies are used restore functional after editing, including efficient single-strand annealing (SSA) homologous recombination in STU-SR-SSA systems. STU-SR-base editor leverage base reinstate start codon, enriching C-to-T A-to-G Our results showcase effectiveness these STU-SR enhancing events monocot rice, encompassing Cas9 nuclease-based targeted mutagenesis, cytosine adenine editing. The exhibit compatibility with variants, such as PAM-less SpRY, shown boost Brassica oleracea, dicot vegetable crop. summary, have developed highly versatile enrichment
Language: Английский
Citations
4
aBIOTECH, Journal Year: 2024, Volume and Issue: 5(2), P. 189 - 195
Published: April 22, 2024
Small mutations in the core promoter region of a gene may result substantial changes expression strengths. However, targeting TA-rich sequences promoters pose challenge for Cas9 variants such as SpCas9 and other G-rich PAM-compatible Cas9s. In this study, we engineered unique FrCas9 system derived from
Language: Английский
Citations
4
Archives of Microbiology, Journal Year: 2024, Volume and Issue: 206(5)
Published: April 23, 2024
Language: Английский
Citations
4
Frontiers in Genome Editing, Journal Year: 2025, Volume and Issue: 6
Published: Jan. 8, 2025
Virus-induced genome editing (VIGE) technologies have been developed to address the limitations plant editing, which heavily relies on genetic transformation and regeneration. However, application of VIGE in plants is hampered by challenge posed size commonly used gene nucleases, Cas9 Cas12a. To overcome this challenge, we employed intein-mediated protein splicing divide SaCas9 transcript into two segments (Split-v1) three (Split-v3). The Split-v1 system demonstrated efficiencies transgenic comparable those achieved with wild-type SaCas9, ranging from 70.2% 96.1%. Additionally, constructed barley stripe mosaic virus (BSMV)-based vectors co-express gRNAs targeting LcHRC, LcGW2, LcTB1 sheepgrass (Leymus chinensis), a Gramineae forage species known for its recalcitrance transformation. Infected leaves exhibited 10.40% 37.03%. These results demonstrate potential split nuclease systems broaden applicability challenging species.
Language: Английский
Citations
0
Plant Biotechnology Journal, Journal Year: 2025, Volume and Issue: unknown
Published: Jan. 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
Language: Английский
Citations
0