Mirza Jaynul Baig

Genome-editing applications in biological organisms rely on a limited set of naturally occurring enzymes. Artificial intelligence (AI) is transforming protein design. Here, building on the AI-designed nuclease OpenCRISPR-1, we establish the plant-optimized AI-designed editors (PAiD) as a versatile genome-editing platform in plants. PAiD supports NHEJ-mediated indel generation, adenine and cytosine base editing, and prime editing, with robust performance across multiple loci comparable to SpCas9. These results highlight the translational potential of AI-designed nucleases for plant genome engineering.

C ₄ photosynthesis evolved through adaptive changes in key metabolic enzymes that enhance carbon fixation efficiency, yet transferring such traits into C ₃ crops has met limited success. Using precision prime editing, we introduced an evolutionarily conserved single amino acid substitution (Arg→Gly) into phosphoenolpyruvate carboxylase (PEPC) in rice. Edited plants exhibited enhanced PEPC activity in the presence of the feedback inhibitor malate, increased chlorophyll content, elevated photosynthetic rates under ambient CO ₂ , and increased seed size and weight. Remarkably, seeds from the edited lines showed simultaneous enrichment of zinc (Zn), iron (Fe), and protein, hereafter referred to as ZiP rice. This work demonstrates that a single, evolutionarily guided residue change can reprogram core carbon metabolism in a C ₃ crop, enabling concurrent improvements in photosynthesis and grain nutritional quality.

Low light intensity is a major abiotic stress that severely affects rice yields, particularly in India and Southeast Asia, causing yield reductions of 35–40% during the wet season compared to the dry season. Tolerant rice genotypes exhibit adaptive changes at anatomical, physiological, biochemical, and molecular levels under low-light stress, enabling higher yields compared to susceptible varieties. Our study identified 20 novel QTLs associated with grain yields and nine related traits under low-light and control (normal)-light conditions, using a recombinant inbred line (RIL) population derived from the cross between the low-light-tolerant variety Swarnaprabha and the low-light-susceptible variety IR8. Across the Kharif seasons of 2019 and 2021, 33 stable QTLs were identified, with 11, 13, and 9 QTLs specific to low-light, normal-light, and both conditions, respectively. Of these, Swarnaprabha contributed 28 QTLs, while five were contributed by IR8. Notably, the study identified 11 and 9 novel QTLs under low-light and both conditions, respectively. Three hotspot regions on chromosomes 1, 4, and 8 were identified. These regions harbored 10 novel QTLs and revealed twenty candidate genes, out of which three key hub genes, OsAUX1, OsSBDCP1, and OsNPF5.16, were identified. These hub genes are involved in hormone signaling, starch metabolism, and nitrogen metabolism, respectively. A comprehensive expression analysis of these genes indicated that they are linked to low-light tolerance, offering deeper insights into the genetic and molecular mechanisms underlying low-light resilience. These findings provide valuable genomic resources and potential markers for breeding programs for improving rice productivity under low-light conditions.

Photosynthetica covers wide range of photosynthesis research or research including photosynthetic parameters of both experimental and theoretical nature.

Key message A long tracrRNA (tracr-L), which naturally act as single guide RNA, and its truncated version, Δtracr-L, from S. pyogenes, efficiently induce Cas9-mediated double-strand breaks (DSBs) in plant genomic loci, as demonstrated by in vitro cleavage assay and protoplast transfection. Abstract CRISPR-Cas system provides a form of immune memory in prokaryotes and archaea, protecting them against viruses and foreign genetic elements. In Streptococcus pyogenes, this system includes the pre-crRNA along with another non-coding RNA, tracrRNA, which aids in CRISPR-based immunity. In S. pyogenes, two distinct tracrRNAs are produced: a long form (tracr-L) and a short form (tracr-S). The tracr-S regulates crRNA biogenesis and Cas9 cleavage, while tracr-L suppresses CRISPR-Cas expression by targeting the Cas9 promoter to prevent autoimmunity. Deleting 79 nucleotides from tracr-L results in Δtracr-L, which retains similar functionality in gene repression. This study investigates, for the first time, the effectiveness of tracr-L, and Δtracr-L in genome editing within plant systems. In vitro cleavage assays using purified Cas9 and synthesized sgRNAs targeting the Cas9 gene, OsPDS, and the OsSWEET11 promoter revealed that across all target sites, tracr-S demonstrated the highest cleavage efficiency compared to tracr-L and Δtracr-L. For in vivo genome editing, we transfected rice protoplasts with tracr-L, Δtracr-L, and tracr-S, targeting three rice genes: OsPDS, OsSPL14, and the promoter of OsSWEET14. Amplicon deep sequencing revealed various types of indels at the target regions across all three tracrRNA versions, indicating comparable levels of efficiency. This study establishes the utility of both the long-form tracrRNA (tracr-L) and its truncated variant (Δtracr-L) in eukaryote genome editing. These two new forms of tracrRNA provide proof of concept and expand the CRISPR-Cas toolkit for plant genome editing applications, and for eukaryotes more broadly.

Identifying and engineering RNA-guided genome editing nucleases for size reduction is a highly coveted pursuit aimed at overcoming various bottlenecks. The size reduction not only facilitates effective delivery but also contributes to the generation of fusion proteins, enabling versatile applications in genome engineering. In this study, we demonstrate high-efficiency genome editing in both monocots and dicots, with average editing rates ranging from 0.63% to 33.58% and reaching up to 69%, using a 408-amino acid-long transposon-associated TnpB. Furthermore, we repurpose a catalytically dead TnpB system for transcriptional activation and base editing. This miniature TnpB, approximately one-third the size of canonical Cas9/Cas12a, emerges as a highly valuable tool for diverse applications in plant genome engineering and gene regulation.