Biology subjects

Mahfouz, M.

Publications and source records attributed to Mahfouz, M..

8 recordsLinked to original sources

bioRxiv2026

High-efficiency, site-specific integration of kilobase-scale DNA into plant genomic safe harbors via PrimeStack editors

Precise, site-specific integration of large DNA sequences into plant genomes is a cornerstone of crop biotechnology and synthetic biology, yet remains constrained by random insertion, inefficient homologous recombination, and gene targeting. Here, we present PrimeStack, a DSB-independent platform that integrates prime editing with the unidirectional large serine integrase Bxb1, leveraging evolved variants for enhanced activity, to achieve the programmable insertion of multigene, multikilobase cargos at predefined genomic safe harbors in rice. Optimized prime editors first install attP landing sites with high fidelity and heritability followed by Bxb1-mediated recombination that generates irreversible integration of genetic information. PrimeStack achieves integration frequencies of approximately 43-46% (as detected by junction-specific PCR in rice calli), with phenotypic neutrality in regenerated plants, comparing favorably with bidirectional Cre-lox systems. We validate its utility by achieving targeted insertion of a carotenoid cassette at an experimentally confirmed genomic safe harbor. PrimeStack delivers a modular, site-specific gene-stacking platform that enables predictable, multigene trait pyramiding and pathway construction at genomic safe harbors, thereby accelerating the development of improved and resilient crop varieties, as well as scalable plant-based biomanufacturing and a powerful chassis for synthetic biology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/718181v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@59c915org.highwire.dtl.DTLVardef@a0ba4corg.highwire.dtl.DTLVardef@26f8d4org.highwire.dtl.DTLVardef@9a3efe_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2025

Physical and functional interactions of the potassium uptake protein HAK5 and the nitrate transporter NPF6.2 is critical for the mineral nutrition of Arabidopsis

Potassium (K+) starvation induces the expression of gene HAK5 encoding a high-affinity K+ uptake protein, but how plants perceive the K+ status and the signaling intermediaries involved in the response remains largely unknown. To identify key regulators of K+ nutrition in Arabidopsis, a genetic screen was performed using an pHAK5:LUC reporter line, and a mutant showing stable induction of the reporter under K+-sufficient conditions was isolated. Mapping-by-sequencing identified two linked mutations affecting genes involved in K+ and nitrate nutrition, namely a loss-of-function in the K+ uptake channel AKT1 and a gain-of-function allele of the nitrate transporter NPF6.2/NRT1.4 (NPF6.2V210M) that doubled the rate of nitrate transport. We report that the physical interaction of NPF6.2 and HAK5 transport proteins resulted in reciprocal interference. Co-expression in Xenopus oocytes of NPF6.2 with the regulatory kinase CIPK23 or the mutant protein NPF6.2V210M alone inhibited HAK5 transport, whereas HAK5 inhibited nitrate transport by NPF6.2 and NPF6.2V210M. We conclude that mutation NPF6.2V210M enhanced the nutritional defects associated to the loss of AKT1 function through the inhibition of HAK5. These findings evidence an intimate molecular crosstalk between transporters involved in the mineral nutrition of plants. The mutual interference when both transport systems are operative may represent a novel integrative regulatory mechanism in mineral nutrition.

plant biology↗

bioRxiv2025

Characterization and engineering of highly efficient Cas12j genome editors

The large size of CRISPR-Cas enzymes limits their delivery for therapeutic applications. Cas12j nucleases offers hypercompact alternative but show moderate editing efficiency. To overcome this limitation, we identified eight novel Cas12j orthologues (Cas12j-11 to Cas12j-18) from viral metagenomes. All showed low editing activity in mammalian cells. We engineered T5 exonuclease-Cas12j fusions (T5Exo-Cas12j), two of which, T5Exo-Cas12j-12, and -18 exhibited up to 42% editing in HEK293T and 9% in K-562 cells, outperforming wild-type Cas12j counterparts and comparable to LbCas12a. Intriguingly, robust in cellula editing in both HEK293T and K-562 cells was strictly dependent on the presence of 5'-TAC trinucleotides within the target DNA sequence. Furthermore, we fused the Cas12j orthologues with the TadA8e deaminase and developed base editors, termed Be-(d)Cas12j. Among these, Be-(d)Cas12j-13 demonstrated efficient A-to-G base conversion in mammalian cells. This study expands the CRISPR toolbox by characterizing and engineering novel Cas12j orthologues into compact, high-efficiency genome editors.

bioengineering↗

bioRxiv2024

Fusions of catalytically inactive RusA to FokI nuclease coupled with PNA enable programable site-specific double-stranded DNA breaks

Programmable site-specific nucleases have revolutionized the genome editing. However, these systems still face challenges such as guide dependency, delivery issues, and off-target effects. Harnessing the natural functions of structure-guided nucleases offer promising alternatives for generating site-specific double-strand DNA breaks. Yet, structure-guided nucleases require precise reaction conditions and validation for in-vivo applicability. To address these limitations, we developed the PNA-Coupled FokI-(d)RusA (PC-FIRA) system. PC-FIRA combines the sequence-specific binding ability of peptide nucleic acids (PNAs) with the catalytic efficiency of FokI nuclease fused to a structurally-guided inactive RusA resolvase (FokI-(d)RusA). This system allows for precise double-strand DNA breaks without the constraints of existing site-specific nuclease and structure-guided nucleases. Through in vitro optimizations, we achieved high target specificity and cleavage efficiency. This included adjusting incubation temperature, buffer composition, ion concentration, and cleavage timing. Diverse DNA structures, such as Holliday Junctions, linear, and circular DNA, were tested demonstrating the potential activity on different target forms. Further investigation has revealed the PC-FIRA system capacity for facilitating the precise deletion of large DNA fragments. This can be useful in cloning, large-fragment DNA assembly, and genome engineering, with promising applications in biotechnology, medicine, agriculture, and synthetic biology.

bioengineering↗

bioRxiv2021

Synthetic evolution of herbicide resistance using a T7 RNAP-based random DNA base editor

Synthetic directed evolution via localized sequence diversification and the simultaneous application of selection pressure is a promising method for producing new, beneficial alleles that affect traits of interest in diverse species; however, this technique has rarely been applied in plants. Developing systems to induce localized sequence diversification at high efficiency will expand our ability to evolve traits of interest that improve global food security. In this study, we designed, built, and tested a chimeric fusion of T7 RNA Polymerase (RNAP) and deaminase to enable the localized sequence diversification of a target sequence of interest. We tested our T7 RNAP-DNA base editor in Nicotiana benthamiana transient assays to target a transgene expressing GFP under the control of the T7 promoter. More than 7% of C nucleotides were converted to T in long segments of the GFP sequence. We then targeted the T7 promoter-driven ACETOLACTATE SYNTHASE (ALS) sequence that had been stably integrated in the rice (Oryza sativa) genome and generated C-to-T and G-to-A transitions. We used herbicide treatment as selection pressure for the evolution of the ALS sequence, resulting in the enrichment of herbicide-responsive residues. We then targeted these herbicide-responsive regions in the rice genome using a CRISPR-directed evolution platform and identified herbicide-resistant ALS variants. Thus, our system could be used for the continuous synthetic evolution of gene functions to produce variants with improved herbicide resistance, as well as for other trait engineering applications.

synthetic biology↗

bioRxiv2018

Engineering Plant Architecture via CRISPR/Cas9-mediated Alteration of Strigolactone Biosynthesis

Precision plant genome engineering holds much promise for targeted improvement of crop traits via unprecedented single-base level control over the genetic material. Strigolactones (SLs) are a key determinant of plant architecture, known for their role in inhibiting shoot branching (tillering). Here, we used CRISPR/Cas9 in rice (Oryza sativa) for targeted disruption of CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), which controls a key step in SL biosynthesis. The ccd7 mutants exhibited a striking increase in tillering, combined with a dwarf phenotype, which could be rescued by application of the synthetic SL analog GR24. Striga germination assays and liquid chromatography-mass spectrometry analysis showed that root exudates of ccd7 mutants were also SL deficient. Taken together, our results show the power of CRISPR/Cas9 for targeted engineering of plant architecture and for elucidating the molecular underpinnings of architecture-related traits.

plant biology↗

bioRxiv2017

Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato

CRISPR/Cas systems confer molecular immunity against phages and conjugative plasmids in prokaryotes. Recently, CRISPR/Cas9 systems have been used to confer interference against eukaryotic viruses. Here, we engineered Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with the CRISPR/Cas9 system to confer immunity against the Tomato yellow leaf curl virus (TYLCV). Targeting the TYLCV genome with Cas9-single guide RNA at the sequences encoding the coat protein (CP) or replicase (Rep) resulted in efficient virus interference, as evidenced by low accumulation of the TYLCV DNA genome in the transgenic plants. The CRISPR/Cas9-based immunity remained active across multiple generations in the N. benthamiana and tomato plants. Together, our results confirmed the efficiency of the CRISPR/Cas9 system for stable engineering of TYLCV resistance in N. benthamiana and tomato, and opens the possibilities of engineering virus resistance against single and multiple infectious viruses in other crops.

plant biology↗

bioRxiv2017

RNA virus interference via CRISPR/Cas13a system in plants

CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants. CRISPR/Cas13a produced interference against green fluorescent protein (GFP) expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. crRNAs targeting the HC-Pro and GFP sequences exhibited better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs. Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses, and for other RNA manipulations in plants.

molecular biology↗