REMOVER-PITCh: microhomology-assisted long-range gene replacement with highly multiplexed CRISPR-Cas9
Left to right: Shu Matsuzaki, Tetsushi Sakuma, Takashi Yamamoto
Gene replacement technology is essential for comparative genomics and humanization of animal models. Recent advancements of CRISPR gene editing tools have enabled site-specific gene modification such as gene knockout and knock-in, while designing and conducting long-range gene replacement is still challenging, especially when sequence homology exists between the endogenous target region and exogenous donor DNA. To tackle this issue, we devised a new technique named REMOVER (REplacement with Multiplex OVERdigestion)-PITCh, in which CRISPR-Cas9 shredded the target genomic region with highly multiplexed guide RNAs to prevent unintended homology-directed repair via the partial homologies between the genome and the donor. By using REMOVER-PITCh with or without a previously established knock-in-enhancing system named LoAD, successful gene replacement was demonstrated with the removal of the long genomic regions (20-200 kb) in human cells. We expect that our REMOVER-PITCh system will open up a new avenue of long-range genetic engineering not only in cultured cells but also in various animal organisms.
Shu Matsuzaki, Tetsushi Sakuma, Takashi Yamamoto. REMOVER-PITCh: microhomology-assisted long-range gene replacement with highly multiplexed CRISPR-Cas9, In Vitro Cellular & Developmental Biology-Animal, 60:697-707, 2024.
Comparative genetic assessment of somatic embryo-and seed-derived plants of two arabica hybrid coffee cultivars using SRAP and SCoT molecular markers and organellar and nuclear genes sequencing
From top left: Dr. Manoj Kumar Mishra and Mr. Arun Kumar Chanabasappa Huded
From bottom left: Dr. Pavankumar Jingade and Dr. B Muniswamy
Coffee is a favorite beverage of millions of people all over the world. Traditionally, coffee is propagated by seed. In recent years, the propagation of plants derived from somatic embryos has become increasingly popular. Since high-frequency somatic embryogenesis is often associated with somaclonal variation, it is imperative to assess the genetic homogeneity of the regenerated plants derived from somatic embryos before large-scale commercial propagation is attempted. In this study, a comparative assessment of genetic uniformity of somatic embryo-derived and seed-derived plants raised from the same mother plant was undertaken in two Coffea arabica F1 hybrids, S.2794 (Hibrido de Timor (HDT) x Geisha) and S.2800 (Bourbon x HDT). The genetic homogeneity was assessed using sequence-related amplified polymorphism (SRAP) and start codon-targeted (SCoT) markers, as well as the sequencing of seven functional genes. The study revealed a remarkable genetic similarity of 93 to 98% between the mother plants and the plants derived from somatic embryos and seeds in both F1 hybrids. The sequence analysis of functional genes confirmed that the large subunit of ribulose-1,5-biphosphate carboxylase and mitochondrial ribosomal protein had the highest sequence similarity. The zinc finger protein gene showed the highest sequence variability, and the pathogenesis-related protein 1A-like gene showed the lowest variability among the five nuclear genes analyzed. This study represents the first comparative analysis of the genetic homogeneity of somatic embryo-derived and seed-derived plants of self-fertilized arabica coffee cultivars. Further, the current study suggests that commercial plantations can be safely established using seeds from superior plants, as the observed genetic variability between seed- and somatic embryo-derived plants is minimal.
Mishra, M.K., Huded, A.K.C., Jingade, P., Muniswamy, B. Comparative genetic assessment of somatic embryo– and seed-derived plants of two arabica hybrid coffee cultivars using SRAP and SCoT molecular markers and organellar and nuclear genes sequencing. In Vitro Cellular & Developmental Biology-Plant, 60: 422–438, 2024. https://doi.org/10.1007/s11627-024-10436-x
Elimination of sugarcane mosaic virus, sugarcane yellow leaf virus and co-infections in sugarcane (Saccharum spp. hybrids) shoot tips via osmo- and cryo-therapy
Meenu Ghal (left), Khethumusa Cele (middle) and Sandy Snyman (right)
Virus elimination using small (<1 mm) excised shoot meristems/ tips has become a well- adopted technique for virus removal, especially in vegetatively propagated or grafted plants. This approach, sometimes used in combination with chemo- and thermo-therapy, has limitations in that the recovery of plants from small meristems is usually low. Cryotherapy, a cryopreservation-based technique, is effective for virus removal from several vegetatively propagated crops, including sugarcane. A novel concept, that of osmotherapy for virus removal in which osmotic dehydration treatments applied during a cryo-protocol but without the freezing step, was introduced to our lab in 2019 when Teresa González-Arnao visited from the Universidad Veracruzana, Veracruz Mexico. This is a suitable alternative treatment for those cultivars that don’t recover well after cryo-treatment. Khethumusa Cele, a PhD student, further refined the protocol and demonstrated that viral co-infections can be eliminated from larger sized (3 mm) meristems, facilitating higher recovery rates than from small, 1mm meristems. Furthermore, no specialized equipment is necessary for either technique, and the benefit of being able to remove unknown viruses is significant to facilitate safer germplasm exchange and generate clean planting material.
Khethumusa H. Cele, Meenu Ghai, Sandra J. Snyman. Elimination of sugarcane mosaic virus, sugarcane yellow leaf virus and co-infections in sugarcane (Saccharum spp. hybrids) shoot tips via osmo- and cryo-therapy. In Vitro Cellular & Developmental Biology-Plant, 60:405-411, 2024.
Genome-wide identification of WUSHEL-related homeobox genes reveals their differential regulation during cold stress and in vitro organogenesis in Royle ex Benth.
Left to Right: Sagar Gupta, Veerbhan Kesarwani, Ravi Shankar. Studio of Computational Biology & Bioinformatics, The Himalayan Centre for High‑throughput Computational Biology, (HiCHiCoB, A BIC supported by DBT, India), Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology (CSIR-IHBT), (HP), India
Left to Right: Jhilmil Nath, Shubham Joshi, Rohit Joshi. Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology (CSIR-IHBT), (HP), India
Picrorhiza kurrooa Royle ex Benth., a member of the Plantaginaceae family, is a remarkable medicinal plant known for its resilience under extreme environmental conditions of Himalayan region. This compelling study delves into plant-specific WUSCHEL-related homeobox (WOX) transcription factor family in Picrorhiza kurrooa. Through a robust genome-wide analysis, we have discovered different WOX genes, revealing their complicated roles during the process of in vitro organogenesis and under cold stress in P. kurrooa. Our findings revealed differential expression patterns of WOX genes during callus proliferation stage and low temperature stress, whereas WUS genes showed differential response during regeneration stage. This suggests that this transcription factor family plays a crucial role during organ differentiation and adaptive response against adverse weather conditions, thus enabling it to withstand harsh environmental vagaries. By elucidating the molecular mechanisms behind WOX gene function, this study not only shed light on P. kurrooa development, resilience and adaptability but also opens new avenues for harnessing these insights in its conservation efforts. Overall, this research provides critical insights into the molecular crosstalk during in vitro organogenesis and stress tolerance mechanism in high-altitude medicinal plants, paving the way towards developing innovative strategies for enhanced resilience under extreme climatic conditions.
Jhilmil Nath, Shubham Joshi, Sagar Gupta, Veerbhan Kesarwani, Ravi Shankar, Rohit Joshi. Genome-wide identification of WUSHEL-related homeobox genes reveals their differential regulation during cold stress and in vitro organogenesis in Picrorhiza kurrooa Royle ex Benth. In Vitro Cellular & Developmental Biology-Plant, 60(4):439-455, 2024.
