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1. Genome- and transcriptome-wide off-target analyses of an improved cytosine base editor

   https://doi.org/10.1093/plphys/kiab264  

   Randall, Linnell Bentley; Sretenovic, Simon; Wu, Yuechao; Yin, Desuo; Zhang, Tao; Van Eck, Joyce; Qi, Yiping (June 2021, Plant Physiology)

   null (Ed.)

   Abstract Cytosine base editors (CBEs) are promising tools for precise genome editing in plants. It is important to investigate potential off-target effects of an efficient CBE at the genome and transcriptome levels in a major crop. Based on comparison of five cytidine deaminases and two different promoters for expressing sgRNAs, we tested a highly efficient A3A/Y130F-BE3 system for efficient C-to-T base editing in tomato (Solanum lycopersicum). We then conducted whole-genome sequencing (WGS) of four base-edited tomato plants, three GFP-expressing control plants, and two wild-type (WT) plants. The sequencing depths ranged from 25X to 49X with read mapping rates above 97%. No sgRNA-dependent off-target mutations were detected. Our data show an average of ∼1000 single nucleotide variations (SNVs) and ∼100 insertions and deletions (indels) per GFP control plant. Base-edited plants had on average elevated levels of SNVs (∼1250) and indels (∼300) per plant. On average, about 200 more C-to-T (G-to-A) mutations were found in a base-edited plant than a GFP control plant, suggesting some level of sgRNA-independent off-target effects, though the difference is not statistically significant. We also conducted RNA sequencing (RNA-seq) of the same four base-edited plants and three GFP control plants. An average of ∼200 RNA SNVs was discovered per plant for either base-edited or GFP control plants. Furthermore, no specific enrichment of C-to-U mutations can be found in the base-edited plants. Hence, we cannot find any evidence for bona fide off-target mutations by A3A/Y130F-BE3 at the transcriptome level. 

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2. Predictable NHEJ insertion and assessment of HDR editing strategies in plants

   https://doi.org/10.3389/fgeed  

   Molla, Kutubuddin A; Shih, J; Wheatley, Matthew S; Yang, Yinong (March 2022, Frontiers in genome editing)

   Canonical CRISPR-Cas9 genome editing technique has profoundly impacted the fields of plant biology, biotechnology, and crop improvement. Since non-homologous end joining (NHEJ) is usually considered to generate random indels, its high efficiency mutation is generally not pertinent to precise editing. Homology-directed repair (HDR) can mediate precise editing with supplied donor DNA, but it suffers from extreme low efficiency in higher plants. Therefore, precision editing in plants will be facilitated by the ability to predict NHEJ repair outcome and to improve HDR efficiency. Here, we report that NHEJ-mediated single nucleotide insertion at different rice genes is predictable based on DNA sequences at the target loci. Three mutation prediction tools (inDelphi, FORECasT, and SPROUT) have been validated in the rice plant system. We also evaluated the chimeric guide RNA (cgRNA) and Cas9-Retron precISe Parallel Editing via homologY (CRISPEY) strategies to facilitate donor template supply for improving HDR efficiency in Nicotiana benthamiana and rice. However, neither cgRNA nor CRISPEY improved plant HDR editing efficiency in this study. Interestingly, our data indicate that tethering of 200–250 nucleotides long sequence to either 5′ or 3′ ends of guide RNA did not significantly affect Cas9 cleavage activity. 

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3. A New Transgenic Tool to Study the Ret Signaling Pathway in the Enteric Nervous System

   https://doi.org/10.3390/ijms232415667  

   Bandla, Ashoka; Melancon, Ellie; Taylor, Charlotte R.; Davidson, Ann E.; Eisen, Judith S.; Ganz, Julia (December 2022, International Journal of Molecular Sciences)

   The receptor tyrosine kinase Ret plays a critical role in regulating enteric nervous system (ENS) development. Ret is important for proliferation, migration, and survival of enteric progenitor cells (EPCs). Ret also promotes neuronal fate, but its role during neuronal differentiation and in the adult ENS is less well understood. Inactivating RET mutations are associated with ENS diseases, e.g., Hirschsprung Disease, in which distal bowel lacks ENS cells. Zebrafish is an established model system for studying ENS development and modeling human ENS diseases. One advantage of the zebrafish model system is that their embryos are transparent, allowing visualization of developmental phenotypes in live animals. However, we lack tools to monitor Ret expression in live zebrafish. Here, we developed a new BAC transgenic line that expresses GFP under the ret promoter. We find that EPCs and the majority of ENS neurons express ret:GFP during ENS development. In the adult ENS, GFP+ neurons are equally present in females and males. In homozygous mutants of ret and sox10—another important ENS developmental regulator gene—GFP+ ENS cells are absent. In summary, we characterize a ret:GFP transgenic line as a new tool to visualize and study the Ret signaling pathway from early development through adulthood. 

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4. Widespread dissolved inorganic carbon-modifying toolkits in genomes of autotrophic Bacteria and Archaea and how they are likely to bridge supply from the environment to demand by autotrophic pathways

   https://doi.org/10.1128/aem.01557-23  

   Scott, Kathleen M.; Payne, Ren R.; Gahramanova, Arin (February 2024, Applied and Environmental Microbiology)

   Bose, Arpita (Ed.)

   ABSTRACT Using dissolved inorganic carbon (DIC) as a major carbon source, as autotrophs do, is complicated by the bedeviling nature of this substance. Autotrophs using the Calvin-Benson-Bassham cycle (CBB) are known to make use of a toolkit comprised of DIC transporters and carbonic anhydrase enzymes (CA) to facilitate DIC fixation. This minireview provides a brief overview of the current understanding of how toolkit function facilitates DIC fixation inCyanobacteriaand someProteobacteriausing the CBB and continues with a survey of the DIC toolkit gene presence in organisms using different versions of the CBB and other autotrophic pathways (reductive citric acid cycle, Wood-Ljungdahl pathway, hydroxypropionate bicycle, hydroxypropionate-hydroxybutyrate cycle, and dicarboxylate-hydroxybutyrate cycle). The potential function of toolkit gene products in these organisms is discussed in terms of CO₂and HCO₃⁻supply from the environment and demand by the autotrophic pathway. The presence of DIC toolkit genes in autotrophic organisms beyond those using the CBB suggests the relevance of DIC metabolism to these organisms and provides a basis for better engineering of these organisms for industrial and agricultural purposes. 

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5. Image‐based root phenotyping links root architecture to micronutrient concentration in cassava

   https://doi.org/10.1002/ppp3.10130  

   Busener, Natalie; Kengkanna, Jitrana; Saengwilai, Patompong Johns; Bucksch, Alexander (July 2020, PLANTS, PEOPLE, PLANET)

   Societal Impact Statement: Micronutrient deficiency or “hidden hunger” is estimated to affect two billion people worldwide and increasing the micronutrient concentration of food could play an important role in tackling this global challenge. Using a combination of imaging techniques and atomic absorption spectroscopy, we describe a link between root phenotype and micronutrient concentration in cassava, which could enable new phenotypic selection strategies for breeding. This approach could be used with existing breeding infrastructure to enhance the micronutrient concentration of cassava and hence, benefit the health of people, particularly in low‐income countries where cassava is consumed as a staple crop. Summary - Cassava storage roots are a staple food in low‐income countries of South‐East Asia and sub‐Saharan Africa, where growth stunting is prevalent as a consequence of micronutrient deficiencies. We aim to link phenotypes of field‐grown cassava roots to micronutrient concentration in the edible storage roots as a simple way to improve phenotypic selection for nutritional value in cassava. - We used existing and newly developed imaging techniques to quantify root phenotypes of the cassava root architecture over time and used flame atomic absorption spectroscopy to measure micronutrient concentration in storage roots. Both together allow the association of root phenotypes with micronutrient concentration in mature cassava roots. We show that early and late bulking genotypes in cassava exhibit distinct foraging behaviors that are associated with micronutrient concentration in the edible storage root. Our observations suggest that late bulking cassava is a key to provide sufficient micronutrients in the edible storage root. - The association between root phenotype and micronutrient concentration with imaging techniques allows phenotypic selection for enhanced micronutrient concentration. Therefore, implementing image‐based phenotyping into cassava breeding programs in sub‐Saharan Africa and South‐East Asia could be an essential element to resolve micronutrient deficiencies that puts individuals at a higher risk of growth stunting. 

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