Biology subjects

Butt, H.

Publications and source records attributed to Butt, H..

4 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↗

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

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↗