Genome- and transcriptome-wide off-target analyses of a high-efficiency adenine base editor in tomato.

Adenine base editors (ABEs) are valuable, precise genome editing tools in plants. In recent years, the highly promising ADENINE BASE EDITOR8e (ABE8e) was reported for efficient A-to-G editing. However, compared to monocots, comprehensive off-target analyses for ABE8e are lacking in dicots. To determine the occurrence of off-target effects in tomato (Solanum lycopersicum), we assessed ABE8e and a high-fidelity version, ABE8e-HF, at 2 independent target sites in protoplasts, as well as stable T0 lines. Since ABE8e demonstrated higher on-target efficiency than ABE8e-HF in tomato protoplasts, we focused on ABE8e for off-target analyses in T0 lines. We conducted whole-genome sequencing (WGS) of wild-type (WT) tomato plants, green fluorescent protein (GFP)-expressing T0 lines, ABE8e-no-gRNA control T0 lines, and edited T0 lines. No guide RNA (gRNA)-dependent off-target edits were detected. Our data showed an average of approximately 1,200 to 1,500 single-nucleotide variations (SNVs) in either GFP control plants or base-edited plants. Also, no specific enrichment of A-to-G mutations were found in base-edited plants. We also conducted RNA sequencing (RNA-seq) of the same 6 base-edited and 3 GFP control T0 plants. On average, approximately 150 RNA-level SNVs were discovered per plant for either base-edited or GFP controls. Furthermore, we did not find enrichment of a TA motif on mutated adenine in the genomes and transcriptomes in base-edited tomato plants, as opposed to the recent discovery in rice (Oryza sativa). Hence, we could not find evidence for genome- and transcriptome-wide off-target effects by ABE8e in tomato.

OsHIPL1, a hedgehog-interacting protein-like 1 protein, increases seed vigour in rice.

The cultivation of rice varieties with high seed vigour is vital for the direct seeding of rice, and the molecular basis of regulation of seed vigour remains elusive. Here, we cloned a new gene OsHIPL1, which encodes hedgehog-interacting protein-like 1 protein as a causal gene of the major QTL qSV3 for rice seed vigour. OsHIPL1 was mainly localized in the plasma membrane and nucleus. RNA sequencing (RNA-seq) revealed that the ABA-related genes were involved in the OsHIPL1 regulation of seed vigour in rice. The higher levels of endogenous ABA were measured in germinating seeds of OsHIPL1 mutants and NIL-qsv3 line compared to IR26 plants, with two up-regulated ABA biosynthesis genes (OsZEP and OsNCED4) and one down-regulated ABA catabolism gene OsABA8ox3. The expression of abscisic acid-insensitive 3 (OsABI3), OsABI4 and OsABI5 was significantly up-regulated in germinating seeds of OsHIPL1 mutants and NIL-qsv3 line compared to IR26 plants. These results indicate that the regulation of seed vigour of OsHIPL1 may be through modulating endogenous ABA levels and altering OsABIs expression during seed germination in rice. Meanwhile, we found that OsHIPL1 interacted with the aquaporin OsPIP1;1, then affected water uptake to promote rice seed germination. Based on analysis of single-nucleotide polymorphism data of rice accessions, we identified a Hap1 haplotype of OsHIPL1 that was positively correlated with seed germination. Our findings showed novel insights into the molecular mechanism of OsHIPL1 on seed vigour.

CRISPR-BETS: a base-editing design tool for generating stop codons.

Cytosine base editors (CBEs) can install a predefined stop codon at the target site, representing a more predictable and neater method for creating genetic knockouts without altering the genome size. Due to the enhanced predictability of the editing outcomes, it is also more efficient to obtain homozygous mutants in the first generation. With the recent advancement of CBEs on improved editing activity, purify and specificity in plants and animals, base editing has become a more appealing technology for generating knockouts. However, there is a lack of design tools that can aid the adoption of CBEs for achieving such a purpose, especially in plants. Here, we developed a user-friendly design tool named CRISPR-BETS (base editing to stop), which helps with guide RNA (gRNA) design for introducing stop codons in the protein-coding genes of interest. We demonstrated in rice and tomato that CRISPR-BETS is easy-to-use, and its generated gRNAs are highly specific and efficient for generating stop codons and obtaining homozygous knockout lines. While we tailored the tool for the plant research community, CRISPR-BETS can also serve non-plant species.

Identification of OsPK5 involved in rice glycolytic metabolism and GA/ABA balance for improving seed germination via genome-wide association study.

Seed germination plays a pivotal role in the plant life cycle, and its precise regulatory mechanisms are not clear. In this study, 19 quantitative trait loci (QTLs) associated with rice seed germination were identified through genome-wide association studies (GWAS) of the following traits in 2016 and 2017: germination rate (GR) at 3, 5, and 7 days after imbibition (DAI) and germination index (GI). Two major stable QTLs, qSG4 and qSG11.1, were found to be associated with GR and GI over 2 continuous years. Furthermore, OsPK5, encoding a pyruvate kinase, was shown to be a crucial regulator of seed germination in rice, and might be a causal gene of the key QTL qSG11.1, on chromosome 11. Natural variation in OsPK5 function altered the activity of pyruvate kinase. The disruption of OsPK5 function resulted in slow germination and seedling growth during seed germination, blocked glycolytic metabolism, caused glucose accumulation, decreased energy levels, and affected the GA/ABA balance. Taken together, our results provide novel insights into the roles of OsPK5 in seed germination, and facilitate its application in rice breeding to improve seed vigour.

A novel pyroptosis-related gene signature predicts the prognosis of glioma through immune infiltration.

BACKGROUND: Glioma is the most common primary intracranial tumour and has a very poor prognosis. Pyroptosis, also known as inflammatory necrosis, is a type of programmed cell death that was discovered in recent years. The expression and role of pyroptosis-related genes in gliomas are still unclear. METHODS: In this study, we analysed the RNA-seq and clinical information of glioma patients from The Cancer Genome Atlas (TCGA) database and Chinese Glioma Genome Atlas (CGGA) database. To investigate the prognosis and immune microenvironment of pyroptosis-related genes in gliomas, we constructed a risk model based on the TCGA cohort. The patients in the CGGA cohort were used as the validation cohort. RESULTS: In this study, we identified 34 pyroptosis-related differentially expressed genes (DEGs) in glioma. By clustering these DEGs, all glioma cases can be divided into two clusters. Survival analysis showed that the overall survival time of Cluster 1 was significantly higher than that of Cluster 2. Using the TCGA cohort as the training set, a 10-gene risk model was constructed through univariate Cox regression analysis and LASSO Cox regression analysis. According to the risk score, gliomas were divided into high-risk and low-risk groups. Survival analysis showed that the low-risk group had a longer survival time than the high-risk group. The above results were verified in the CGGA validation cohort. To verify that the risk model was independent of other clinical features, the distribution and the Kaplan-Meier survival curves associated with risk scores were performed. Combined with the characteristics of the clinical cases, the risk score was found to be an independent factor predicting the overall survival of patients with glioma. The analysis of single sample Gene Set Enrichment Analysis (ssGSEA) showed that compared with the low-risk group, the high-risk group had immune cell and immune pathway activities that were significantly upregulated. CONCLUSION: We established 10 pyroptosis-related gene markers that can be used as independent clinical predictors and provide a potential mechanism for the treatment of glioma.

A quantitative trait locus, qSE3, promotes seed germination and seedling establishment under salinity stress in rice.

Seed germination is a complex trait determined by both quantitative trait loci (QTLs) and environmental factors and also their interactions. In this study, we mapped one major QTLqSE3 for seed germination and seedling establishment under salinity stress in rice. To understand the molecular basis of this QTL, we isolated qSE3 by map-based cloning and found that it encodes a K+ transporter gene, OsHAK21. The expression of qSE3 was significantly upregulated by salinity stress in germinating seeds. Physiological analysis suggested that qSE3 significantly increased K+ and Na+ uptake in germinating seeds under salinity stress, resulting in increased abscisic acid (ABA) biosynthesis and activated ABA signaling responses. Furthermore, qSE3 significantly decreased the H2 O2 level in germinating seeds under salinity stress. All of these seed physiological changes modulated by qSE3 might contribute to seed germination and seedling establishment under salinity stress. Based on analysis of single-nucleotide polymorphism data of rice accessions, we identified a HAP3 haplotype of qSE3 that was positively correlated with seed germination under salinity stress. This study provides important insights into the roles of qSE3 in seed germination and seedling establishment under salinity stress and facilitates the practical use of qSE3 in rice breeding.

Influence of isopropylmalate synthase OsIPMS1 on seed vigour associated with amino acid and energy metabolism in rice.

Seed vigour is an imperative trait for the direct seeding of rice. Isopropylmalate synthase (IPMS) catalyses the committed step of leucine (Leu) biosynthesis, but its effect on seed vigour remains unclear. In this study, rice OsIPMS1 and OsIPMS2 was cloned, and the roles of OsIPMS1 in seed vigour were mainly investigated. OsIPMS1 and OsIPMS2 catalyse Leu biosynthesis, and Leu feedback inhibits their IPMS activities. Disruption of OsIPMS1 resulted in low seed vigour under various conditions, which might be tightly associated with the reduction of amino acids in germinating seeds. Eleven amino acids that associated with stress tolerance, GA biosynthesis and tricarboxylic acid (TCA) cycle were significantly reduced in osipms1 mutants compared with those in wide type (WT) during seed germination. Transcriptome analysis indicated that a total of 1209 differentially expressed genes (DEGs) were altered in osipms1a mutant compared with WT at the early germination stage, wherein most of the genes were involved in glycolysis/gluconeogenesis, protein processing, pyruvate, carbon, fructose and mannose metabolism. Further analysis confirmed that the regulation of OsIPMS1 in seed vigour involved in starch hydrolysis, glycolytic activity and energy levels in germinating seeds. The effects of seed priming were tightly associated with the mRNA levels of OsIPMS1 in priming seeds. The OsIPMS1 might be used as a biomarker to determine the best stop time-point of seed priming in rice. This study provides novel insights into the function of OsIPMS1 on seed vigour and should have practical applications in seed priming of rice.

Genome editing in rice and tomato with a small Un1Cas12f1 nuclease.

The clustered regularly interspaced short palindromic repeats (CRISPR) systems have been demonstrated to be the foremost compelling genetic tools for manipulating prokaryotic and eukaryotic genomes. Despite the robustness and versatility of Cas9 and Cas12a/b nucleases in mammalian cells and plants, their large protein sizes may hinder downstream applications. Therefore, investigating compact CRISPR nucleases will unlock numerous genome editing and delivery challenges that constrain genetic engineering and crop development. In this study, we assessed the archaeal miniature Un1Cas12f1 type-V CRISPR nuclease for genome editing in rice and tomato protoplasts. By adopting the reengineered guide RNA modifications ge4.1 and comparing polymerase II (Pol II) and polymerase III (Pol III) promoters, we demonstrated uncultured archaeon Cas12f1 (Un1Cas12f1) genome editing efficacy in rice and tomato protoplasts. We characterized the protospacer adjacent motif (PAM) requirements and mutation profiles of Un1Cas12f1 in both plant species. Interestingly, we found that Pol III promoters, not Pol II promoters, led to higher genome editing efficiency when they were used to drive guide RNA expression. Unlike in mammalian cells, the engineered Un1Cas12f1-RRA variant did not perform better than the wild-type Un1Cas12f1 nuclease, suggesting continued protein engineering and other innovative approaches are needed to further improve Un1Cas12f1 genome editing in plants.

High performance TadA-8e derived cytosine and dual base editors with undetectable off-target effects in plants.

Cytosine base editors (CBEs) and adenine base editors (ABEs) enable precise C-to-T and A-to-G edits. Recently, ABE8e, derived from TadA-8e, enhances A-to-G edits in mammalian cells and plants. Interestingly, TadA-8e can also be evolved to confer C-to-T editing. This study compares engineered CBEs derived from TadA-8e in rice and tomato cells, identifying TadCBEa, TadCBEd, and TadCBEd_V106W as efficient CBEs with high purity and a narrow editing window. A dual base editor, TadDE, promotes simultaneous C-to-T and A-to-G editing. Multiplexed base editing with TadCBEa and TadDE is demonstrated in transgenic rice, with no off-target effects detected by whole genome and transcriptome sequencing, indicating high specificity. Finally, two crop engineering applications using TadDE are shown: introducing herbicide resistance alleles in OsALS and creating synonymous mutations in OsSPL14 to resist OsMIR156-mediated degradation. Together, this study presents TadA-8e derived CBEs and a dual base editor as valuable additions to the plant editing toolbox.

On- and Off-Target Analyses of CRISPR-Cas12b Genome Editing Systems in Rice.

The CRISPR-associated Cas12b system is the third most efficient CRISPR tool for targeted genome editing in plants after Cas9 and Cas12a. Although the genome editing ability of AaCas12b has been previously investigated in rice, its off-target effects in plants are largely not known. In this study, we first engineered single-guide RNA (sgRNA) complexes with various RNA scaffolds to enhance editing frequency. We targeted EPIDERMAL PATTERNING FACTOR LIKE 9 (OsEPFL9) and GRAIN SIZE 3 (OsGS3) genes with GTTG and ATTC protospacer adjacent motifs, respectively. The use of two Alicyclobacillus acidoterrestris scaffolds (Aac and Aa1.2) significantly increased the frequency of targeted mutagenesis. Next, we performed whole-genome sequencing (WGS) of stably transformed T0 rice plants to assess off-target mutations. WGS analysis revealed background mutations in both coding and noncoding regions with no evidence of sgRNA-dependent off-target activity in edited genomes. We also showed Mendelian segregation of insertion and deletion (indel) mutations in T1 generation. In conclusion, both Aac and Aa1.2 scaffolds provided precise and heritable genome editing in rice.

Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants.

BACKGROUND: Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants. RESULTS: To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis in E. coli and identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency in T0 plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites. CONCLUSIONS: Our results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.

G-protein Coupled Receptor 34 Promotes Gliomagenesis by Inducing Proliferation and Malignant Phenotype via TGF-Beta/Smad Signaling Pathway.

Background: G-protein coupled receptor 34 (GPR34) is involved in cell motility, differentiation, and mitosis. GPR34 was reported to be highly expressed and play an oncogenic role in several solid tumors. Here, we investigated the mechanisms underlying how GPR34 promotes glioma progression. Methods: Bioinformatic analysis was performed on RNA-seq and clinical data from the gene expression omnibus (GEO), cancer genome atlas (TCGA), and Genotype-Tissue Expression (GTEx) databases. TIMER database and single-sample GSEA (ssGAEA) method were used to investigate the association between the GPR34 expression and immune infiltration level in glioma. Cox regression analysis was employed to ascertain whether the risk signature was an independent prognostic indicator for glioma. The viability and migratory/invasive potential of glioma cells were assessed using Cell Counting Kit-8, colony formation, wound healing, and Transwell assays. Results: We found that GPR34 expression was positively correlated with immune infiltration level and that high GPR34 level may be associated with poor prognosis in glioma. We further found that GPR34 may serve as an independent prognostic marker and prediction factor for the clinicopathological features of glioma. We showed that knocking down GPR34 attenuated the viability and migratory/invasive capacity of glioma cells (U251 and LN229), while GPR34 overexpression exerted the opposite effects. Additionally, core enrichment in the GSEA analysis indicated that GPR34-mediated gliomagenesis was associated with the cell cycle arrest, epithelial-mesenchymal transition (EMT), and activation of the TGF-ß/Smad pathway; furthermore, inhibiting TGF-ß/Smad signaling using LY2157299, a TGF-ß inhibitor, reversed the oncogenic effects and malignant phenotype associated with GPR34 overexpression. Conclusion: GPR34 enhances the malignancy and carcinogenesis of glioma by promoting an EMT-like process, G1/S phase cell cycle transition, and TGF-ß/Smad signaling. Accordingly, GPR34 likely functions as an oncogene in glioma and may represent a potential therapeutic target for this cancer.

Expanding the targeting scope of FokI-dCas nuclease systems with SpRY and Mb2Cas12a.

CRISPR-Cas9 and Cas12a are widely used sequence-specific nucleases (SSNs) for genome editing. The nuclease domains of Cas proteins can induce DNA double strand breaks upon RNA guided DNA targeting. Zinc finger nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have been popular SSNs prior to CRISPR. Both ZFNs and TALENs are based on reconstitution of two monomers with each consisting of a DNA binding domain and a FokI nuclease domain. Inspired by the configuration of ZFNs and TALENs, dimeric FokI-dCas9 systems were previously demonstrated in human cells. Such configuration, based on a pair of guide RNAs (gRNAs), offers great improvement on targeting specificity. To expand the targeting scope of dimeric FokI-dCas systems, the PAM (protospacer adjacent motif)-less SpRY Cas9 variant and the PAM-relaxed Mb2Cas12a system were explored. Rice cells showed that FokI-dSpRY had more robust editing efficiency than a paired SpRY nickase system. Furthermore, a dimeric FokI-dMb2Cas12a system was developed that displayed comparable editing activity to Mb2Cas12a nuclease in rice cells. Finally, a single-chain FokI-FokI-dMb2Cas12a system was developed that cuts DNA outside its targeting sequence, which could be useful for many versatile applications. Together, this work greatly expanded the FokI based CRISPR-Cas systems for genome editing.

Boosting plant genome editing with a versatile CRISPR-Combo system.

CRISPR-Cas9, its derived base editors and CRISPR activation systems have greatly aided genome engineering in plants. However, these systems are mostly used separately, leaving their combinational potential largely untapped. Here we develop a versatile CRISPR-Combo platform, based on a single Cas9 protein, for simultaneous genome editing (targeted mutagenesis or base editing) and gene activation in plants. We showcase the powerful applications of CRISPR-Combo for boosting plant genome editing. First, CRISPR-Combo is used to shorten the plant life cycle and reduce the efforts in screening transgene-free genome-edited plants by activation of a florigen gene in Arabidopsis. Next, we demonstrate accelerated regeneration and propagation of genome-edited plants by activation of morphogenic genes in poplar. Furthermore, we apply CRISPR-Combo to achieve rice regeneration without exogenous plant hormones, which is established as a new method to predominately enrich heritable targeted mutations. In conclusion, CRISPR-Combo is a versatile genome engineering tool with promising applications in crop breeding.

Highly Efficient Genome Editing in Plant Protoplasts by Ribonucleoprotein Delivery of CRISPR-Cas12a Nucleases.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) mediated genome editing is a powerful approach for crop improvement. Traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects. CRISPR delivered as ribonucleoproteins (RNPs) can prevent exogenous DNA integration, minimize off-target effects, and reduce cellular toxicity. Although RNP delivered CRISPR genome editing has been demonstrated in many plant species, optimization strategies that yield high editing efficiencies have not been thoroughly investigated. Using rice and citrus protoplast systems we demonstrated highly efficient genome editing using Cas12a delivered as RNPs. Four Cas12a variants, including LbCas12a, LbCas12a-E795L, AsCas12a, and AsCas12a Ultra, were investigated. Nearly 100% editing efficiency was observed for three out of four target sites by LbCas12a, LbCas12a-E795L, and AsCas12a Ultra, as measured by restriction fragment length polymorphism (RFLP) and verified by next generation sequencing of PCR amplicons. RNP delivery resulted in higher editing efficiencies than plasmid delivery at 32°C and 25°C. LbCas12a and LbCas12a-E795L demonstrated increased editing efficiencies in comparison to AsCas12a and AsCas12a Ultra, especially when used at lower RNP concentrations. In addition, we discovered that a 1:1 Cas12a:crRNA molar ratio is sufficient to achieve efficient genome editing. Nuclear localization signals (NLSs) are essential for efficient RNP-based genome editing. However, the different crRNA modifications tested did not significantly improve genome editing efficiency. Finally, we applied the Cas12a RNP system in citrus protoplasts and obtained similarly high editing efficiencies at the target site. Our study provides a comprehensive guideline for Cas12a-mediated genome editing using RNP delivery in plant cells, setting the foundation for the generation of transgene-free genome edited plants.

CUX1 Facilitates the Development of Oncogenic Properties Via Activating Wnt/ß-Catenin Signaling Pathway in Glioma.

Background: Homeobox cut like 1 (CUX1), which often presents aberrated expression in many cancer cells, exerts a crucial role in tumorigenesis. Evidence describing CUX1 in gliomagenesis is scarce, and the effects of CUX1 on the Wnt/ß-catenin pathway have not been reported. Our study aimed to explore the biological functions and molecular mechanisms involved in CUX1 activity in glioma. Methods: Datasets for bioinformatics analysis were obtained from the GEO, TCGA, CGGA, GTEX and CCLE databases. qRT-PCR, western blotting (WB), and immunohistochemistry (IHC) assays were used to investigate the expression patterns of CUX1 among glioma and brain tissues. CUX1 knockdown and overexpression vectors were transfected into glioma cell lines, the CCK-8, clone formation assay, wound healing, Transwell assay, and flow cytometry were performed to detect changes in cell viability, invasiveness, and the cell cycle. WB and immunofluorescence (IF) assays were used to explore changes in cell cycle-related and Wnt/ß-catenin signaling protein levels. Results: Overexpression of CUX1 was identified in glioma tissues, and especially in glioblastoma (GBM), when compared to normal controls and correlated with poor prognosis. In comparison with untreated cells, TJ905 glioma cells overexpressing CUX1 showed higher proliferation and invasion abilities and S phase cell-cycle arrest, while the knockdown of CUX1 suppressed cell invasive ability and induced G1 phase arrest. Active Wnt/ß-catenin signaling was enriched and clustered in a CUX1-associated GSEA/GSVA analysis. IF and WB assays indicated that CUX1 regulated the distribution of Axin2/ß-catenin in glioma cells and regulated the expression of proteins downstream of the Wnt/ß-catenin signaling pathway, suggesting that CUX1 served as an upstream positive regulator of the Wnt/ß-catenin pathway. Finally, the knockdown of Axin2 or ß-catenin could reverse the tumor-promoting effects caused by CUX1 overexpression, suggesting that CUX1 induced gliomagenesis and malignant phenotype by activating the Wnt/ß-catenin signaling pathway. Conclusion: Our data suggested that the transcription factor CUX1 could be a novel therapeutic target for glioma with gene therapy.

Exploring C-To-G Base Editing in Rice, Tomato, and Poplar.

As a precise genome editing technology, base editing is broadly used in both basic and applied plant research. Cytosine base editors (CBEs) and adenine base editors (ABEs) represent the two commonly used base editor types that mediate C-to-T and A-to-G base transition changes at the target sites, respectively. To date, no transversion base editors have been described in plants. Here, we assessed three C-to-G base editors (CGBEs) for targeting sequences with SpCas9's canonical NGG protospacer adjacent motifs (PAMs) as well as three PAM-less SpRY-based CGBEs for targeting sequences with relaxed PAM requirements. The analyses in rice and tomato protoplasts showed that these CGBEs could make C-to-G conversions at the target sites, and they preferentially edited the C6 position in the 20-nucleotide target sequence. C-to-T edits, insertions and deletions (indels) were major byproducts induced by these CGBEs in the protoplast systems. Further assessment of these CGBEs in stably transformed rice and poplar plants revealed the preference for editing of non-GC sites, and C-to-T edits are major byproducts. Successful C-to-G editing in stably transgenic rice plants was achieved by rXRCC1-based CGBEs with monoallelic editing efficiencies up to 38% in T0 lines. The UNG-rAPOBEC1 (R33A)-based CGBE resulted in successful C-to-G editing in polar, with monoallelic editing efficiencies up to 6.25% in T0 lines. Overall, this study revealed that different CGBEs have different preference on preferred editing sequence context, which could be influenced by cell cycles, DNA repair pathways, and plant species.

CRISPR-Act3.0 for highly efficient multiplexed gene activation in plants.

RNA-guided CRISPR activation (CRISPRa) systems have been developed in plants. However, the simultaneous activation of multiple genes remains challenging. Here, we develop a highly robust CRISPRa system working in rice, Arabidopsis and tomato, CRISPR-Act3.0, through systematically exploring different effector recruitment strategies and various transcription activators based on deactivated Streptococcus pyogenes Cas9 (dSpCas9). The CRISPR-Act3.0 system results in fourfold to sixfold higher activation than the state-of-the-art CRISPRa systems. We further develop a tRNA-gR2.0 (single guide RNA 2.0) expression system enabling CRISPR-Act3.0-based robust activation of up to seven genes for metabolic engineering in rice. In addition, CRISPR-Act3.0 allows the simultaneous modification of multiple traits in Arabidopsis, which are stably transmitted to the T3 generations. On the basis of CRISPR-Act3.0, we elucidate guide RNA targeting rules for effective transcriptional activation. To target T-rich protospacer adjacent motifs (PAMs), we transfer this activation strategy to CRISPR-dCas12b and further improve the dAaCas12b-based CRISPRa system. Moreover, we develop a potent near-PAM-less CRISPR-Act3.0 system on the basis of the SpRY dCas9 variant, which outperforms the dCas9-NG system in both activation potency and targeting scope. Altogether, our study has substantially improved the CRISPRa technology in plants and provided plant researchers a powerful toolbox for efficient gene activation in foundational and translational research.

TROAP regulates cell cycle and promotes tumor progression through Wnt/ß-Catenin signaling pathway in glioma cells.

AIMS: Experimental evidence demonstrated a crucial role of TROAP (Trophinin-associated protein) in regulating the cell proliferation of multiple tumors, while TROAP expression and function were largely unknown in glioma. We aimed to investigate the oncogenic role of TROAP and its potential mechanisms in gliomagenesis. METHODS: Four gene expression databases (GEO, TCGA, GTEx and CCLE) were enrolled in our study and used for TROAP expression and survival analysis. TROAP expression was quantified by qRT-PCR, western blot and immunohistochemistry assays in glioma tissues and cell lines. TROAP knockdown and overexpression vector were constructed and transfected into glioma cells. CCK-8, colony formation, transwell, and wound healing assays were used to evaluate cell viability, migration and invasion, flow cytometry to determine cell cycle arrest. Gene set enrichment analysis (GSEA) was conducted to screen the pathway involved in TROAP-high phenotype. The expression of cell cycle and Wnt/ß-Catenin signaling proteins were analyzed by immunofluorescence and western blot. RESULTS: Based on the bioinformatic analysis and a series of functional assays, we found the TROAP was enriched in glioma tissues and cell lines, its overexpression was correlated with the clinicopathologic characteristics and poor prognosis. TROAP knockdown inhibited cell proliferation, migration, invasion, and G1/S cell cycle arrest compared with control group in glioma. Mechanism analysis revealed that TROAP activated Wnt/ß-Catenin pathway and upregulated its downstream targets expression, while silencing ß-Catenin or Axin2 could reverse the tumor-promoting effects caused by TROAP, confirming that TROAP-induced malignant phenotype and tumorigenesis via Wnt/ß-Catenin signaling pathway. CONCLUSION: The present study found that TROAP accelerated the progression of gliomagenesis through Wnt/ß-Catenin pathway, and TROAP might be considered as a novel target for glioma therapy.

PAM-less plant genome editing using a CRISPR-SpRY toolbox.

The rapid development of the CRISPR-Cas9, -Cas12a and -Cas12b genome editing systems has greatly fuelled basic and translational plant research1-6. DNA targeting by these Cas nucleases is restricted by their preferred protospacer adjacent motifs (PAMs). The PAM requirement for the most popular Streptococcus pyogenes Cas9 (SpCas9) is NGG (N = A, T, C, G)7, limiting its targeting scope to GC-rich regions. Here, we demonstrate genome editing at relaxed PAM sites in rice (a monocot) and the Dahurian larch (a coniferous tree), using an engineered SpRY Cas9 variant8. Highly efficient targeted mutagenesis can be readily achieved by SpRY at relaxed PAM sites in the Dahurian larch protoplasts and in rice transgenic lines through non-homologous end joining (NHEJ). Furthermore, an SpRY-based cytosine base editor was developed and demonstrated by directed evolution of new herbicide resistant OsALS alleles in rice. Similarly, a highly active SpRY adenine base editor was developed based on ABE8e (ref. 9) and SpRY-ABE8e was able to target relaxed PAM sites in rice plants, achieving up to 79% editing efficiency with high product purity. Thus, the SpRY toolbox breaks a PAM restriction barrier in plant genome engineering by enabling DNA editing in a PAM-less fashion. Evidence was also provided for secondary off-target effects by de novo generated single guide RNAs (sgRNAs) due to SpRY-mediated transfer DNA self-editing, which calls for more sophisticated programmes for designing highly specific sgRNAs when implementing the SpRY genome editing toolbox.