Aug 2026· Insect Biochemistry and Molecular Biology· Vol 195, pp.
104649
· 0 citations· 38 references
Medicine
TL;DR
The genetic basis of a natural orange-eye mutation in N. lugens is identified and multiple-sgRNA editing is established as an efficient strategy for rapid functional screening in insects.
Abstract
Eye-color mutants provide easily distinguishable visual markers for genetic analysis and genome editing, yet the molecular basis of naturally occurring eye-color variation in Nilaparvata lugens remains unclear. Here, we combined classical genetic analysis, candidate-gene screening, and CRISPR/Cas9 genome editing to elucidate the genetic basis of a stably inherited orange-eye phenotype and evaluate the editing performance of different sgRNA strategies. The orange-eye phenotype was inherited as a single autosomal recessive trait, and the mutant strain showed only limited fitness differences from the WT strain. A 1,287-bp insertion spanning exon 13 and the adjacent intronic region of Nlscarlet introduced a premature stop codon, truncating NlScarlet after the fifth transmembrane helix. The insertion completely cosegregated with the orange-eye phenotype, and CRISPR/Cas9-mediated disruption of Nlscarlet recapitulated the phenotype, demonstrating that this insertion underlies the natural mutation. A separate in-frame deletion of lysine 535 produced a dark-orange-eye phenotype, and structural modeling suggested that this residue may contribute to the interaction between the NlWhite/NlScarlet transporter complex and 3-hydroxykynurenine. Using Nlscarlet as a visually scorable marker, two multiple-sgRNA strategies, each using four sgRNAs, increased the proportions of G0 individuals with complete loss-of-function phenotypes from 30.97% to 84.73% and 92.47%, respectively, and of mutant-eyed G1 progeny from 37.33% to 81.33% and 90.67%, respectively. Heritable mutations generated by both strategies were predominantly large deletions. These findings identify the genetic basis of a natural orange-eye mutation in N. lugens and establish multiple-sgRNA editing as an efficient strategy for rapid functional screening in insects.
A multiplex CRISPR/Cas12a system is established in eggplant accession MEL3, representing, to the authors' knowledge, the first application of this nuclease for genome editing in eggplant and demonstrating the potential of Cas12a for functional genomics, allele engineering, and precision breeding in eggplant.
Marina Martínez-López, Andrea Solana, Andrea Arrones et al.· bioRxiv· 0 citations
CRISPR-SWITCH is presented, a genome engineering strategy that enables deliberate monoallelic editing by exploiting allele-specific CRISPR targeting and can enforce heterozygosity at endogenous loci and enable the generation of viable mammalian models for dominant-negative and dosage-sensitive genetic disorders.
Samantha Norris, Sai Goutham Reddy Yeddula, E. Su et al.· Frontiers in Genome Editing· 0 citations
Nesidiocoris tenuis is an important zoophytophagous mirid bug used as a biological control agent in agriculture, and breeding efforts based on genomic information aim to increase its utility. Visible eye-color mutants are useful genetic markers because they are easily distinguishable and are therefore widely used in insect genetics and genome editing studies. Here, we investigated the genetic basis of a spontaneous red-eye mutant identified in a laboratory strain of N. tenuis. Classical crossing experiments suggested that the red-eye phenotype is controlled by a single recessive locus. RNA-seq and RNA interference (RNAi) analyses identified scarlet and cinnabar as the primary candidate genes associated with the phenotype. Further genomic analysis revealed a large deletion and insertion within exon 5 of the mutant scarlet allele, potentially causing exon skipping and disrupting transporter structure. The insertion pattern is consistent with a microhomology-mediated break-induced replication (MMBIR)/fork stalling and template switching (FoSTeS)-like event that may have been generated through polymerase θ-mediated repair. Together, these findings identify the causative mutation underlying the red-eye phenotype and provide a useful visible marker for future functional genetic studies and genome-assisted breeding in N. tenuis.
Tomofumi Shibata, Kaoru Saeki, C. Saito et al.· bioRxiv· 0 citations
This study uncovers a rare case in which a deletion of just two amino acids is sufficient to generate a potent dominant-negative regulator, designated SmMYB1alf-D, which enables reliable prediction of fruit color and provides a breeding strategy to precisely manipulate anthocyanin metabolism.
Yan Li, Yiwen Tian, Wanyue Li et al.· Plant Communications· 0 citations
Spontaneous mutations are a rare but important source of novel genetic variation, yet their detection and characterization within active breeding programs are seldom documented at gene-level resolution. Grain size and shape are key determinants of rice quality, yield, and market classification. Here, we report the discovery and genetic characterization of a spontaneous short-grain (SG) mutation arising in the long-grain wild-type (WT) advanced breeding line RU2002174 from the LSU AgCenter Rice Breeding Program. The SG phenotype was first observed in 2019 and segregated in subsequent generations as a single recessive gene across both indica and japonica genetic backgrounds. Genetic mapping localized the mutation to a 41.6 kb interval on chromosome 5. Whole-genome sequencing identified a single candidate causal variant: a G→T transversion in exon 4 of SRS3 (Os05g06280), introducing a premature stop codon and resulting in a truncated protein. This allele was absent from representative U.S. breeding germplasm and the IRRI 3K SNP database, demonstrating that it represents a novel spontaneous loss-of-function allele of a previously characterized grain-size gene. These findings document the real-time emergence of functional genetic variation in elite rice germplasm and highlight the importance of monitoring off-types during seed increase and purification in breeding programs. They also provide additional insight into the role of kinesin-mediated cell elongation in determining rice grain architecture.
Maria Montiel, Brijesh Angira, Jonathan K. Richards et al.· bioRxiv· 0 citations
GOLDEN2-like (GLK) transcription factors are key regulators of chloroplast differentiation and photosynthetic gene expression. The classical yellow mutation in Japanese morning glory (Ipomoea nil) produces yellowish-green leaves, whereas an unstable allele, yellow-mutable, produces green somatic sectors on a yellowish-green background. The gene responsible for these mutations was identified as InGLK, which encodes a GOLDEN2-like transcription factor. The stable yellow mutant carried a 4-bp frameshift insertion in InGLK, whereas two yellow-mutable lines carried the Tpn1-family transposon Tpn12 in intron 5. Excision of Tpn12 in germinal revertants left short footprints and restored the green leaf phenotype. Genome searches identified InGLK as the sole GLK gene in I. nil. Pigment analysis of green somatic reversion sectors and yellowish-green background areas showed that most of the measured photosynthetic pigments were significantly reduced in the yellowish-green background, whereas the chlorophyll a/b ratio was unchanged. Chloroplasts in the yellowish-green tissue retained thylakoid-like membranes and starch granule-like structures but had less distinct grana-like stacks and sparse stromal lamellae-like structures. Wild-type-like chloroplast ultrastructure was restored in germinal revertants. These findings show that loss of function of a single-copy GLK gene broadly reduces photosynthetic pigment accumulation and alters chloroplast internal membrane organization. The yellow mutants of I. nil therefore provide a genetic system for examining non-redundant GLK function.
Hibika Umehara, K. Takagi, Soya Nakagawa et al.· bioRxiv· 0 citations