A protocol for the detection of precision genome editing in human cells using One-pot DTECT.

Prime editing is a highly versatile CRISPR-based genome editing technology that allows for the precise installation of desired genetic variants. This protocol describes how to use One-pot DTECT to assess prime editing efficiency in human cells. Key steps include conducting prime editing, extracting genomic DNA, performing AcuI-tagging PCR, capturing genetic signatures, and detecting captured signatures through qualitative, quantitative, and visual methods. One-pot DTECT enables same-day detection of targeted genetic signatures introduced by precision genome editing technologies using off-the-shelf reagents. For complete details on the use and execution of this protocol, please refer to Baudrier et al.1.

One-pot DTECT enables rapid and efficient capture of genetic signatures for precision genome editing and clinical diagnostics.

The detection of genomic sequences and their alterations is crucial for basic research and clinical diagnostics. However, current methodologies are costly and time-consuming and require outsourcing sample preparation, processing, and analysis to genomic companies. Here, we establish One-pot DTECT, a platform that expedites the detection of genetic signatures, only requiring a short incubation of a PCR product in an optimized one-pot mixture. One-pot DTECT enables qualitative, quantitative, and visual detection of biologically relevant variants, such as cancer mutations, and nucleotide changes introduced by prime editing and base editing into cancer cells and human primary T cells. Notably, One-pot DTECT achieves quantification accuracy for targeted genetic signatures comparable with Sanger and next-generation sequencing. Furthermore, its effectiveness as a diagnostic platform is demonstrated by successfully detecting sickle cell variants in blood and saliva samples. Altogether, One-pot DTECT offers an efficient, versatile, adaptable, and cost-effective alternative to traditional methods for detecting genomic signatures.

Species Diversity and Phylogenetic Relationships of Olive Lace Bugs (Hemiptera: Tingidae) Found in South Africa.

Olive lace bugs (Hemiptera: Tingidae) are small sap-sucking insects that feed on wild and cultivated Olea europaea. The diversity of olive lace bug species in South Africa, the most important olive producer on the continent, has been incompletely surveyed. Adult specimens were collected in the Western Cape province for morphological and DNA-based species identification, and sequencing of complete mitogenomes. Cysteochila lineata, Plerochila australis, Neoplerochila paliatseasi and Neoplerochila sp. were found at 12 sites. Intra- and interspecific genetic divergences and phylogenetic clustering in 30 species in 18 genera of Tingidae using new and publicly available DNA barcodes showed high levels of congruity between taxonomic and genetic data. The phylogenetic position of the four species found in South Africa was inferred using new and available mitogenomes of Tingidae. Notably, olive lace bugs formed a cluster of closely related species. However, Cysteochila was non-monophyletic as C. lineata was recovered as a sister species to P. australis whereas Cysteochila chiniana, the other representative of the genus, was grouped with Trachypeplus jacobsoni and Tingis cardui in a different cluster. This result suggests that feeding on O. europaea may have a common origin in Tingidae and warrants future research on potential evolutionary adaptations of olive lace bugs to this plant host.

Mitochondrial genomes and polymorphic regions of Gonimbrasia belina and Gynanisa maja (Lepidoptera: Saturniidae), two important edible caterpillars of Southern Africa.

Mopane worms are the vernacular designation for the edible caterpillars of the African emperor moths Gonimbrasia belina and Gynanisa maja. Both species, particularly G. belina, are widely harvested in Southern Africa, and their populations are declining. Despite their commercial, nutritional, and cultural importance, their genetic data are currently unavailable. We sequenced two complete mitogenomes from each species using Ion Torrent technology, and identified informative markers in the complete mitogenomes of the two species for use in future studies. Comparing the conspecific mitogenomes allowed the identification of regions with high nucleotide diversity in ATP6, ND1, ND4, ND5, ND6, and CYTB genes. The final panels of markers will allow for the survey of 3117 bp in G. belina, and 3990 bp in Gy. maja. Phylogenetic reconstruction within the family Saturniidae recovered the tribe Bunaeini as monophyletic and basal to Saturniidae, and the tribe Attacini as a monophyletic clade nested within the tribe Saturniini. The G. belina and Gy. maja mitogenomes are the first representatives of African Saturniidae, a taxonomic group with relevance as a food resource on the continent. This study represents the first step towards assessing the genetic diversity, population structure, and phylogeography of African edible caterpillars.

First report of the lace bug Neoplerochila paliatseasi (Rodrigues, 1981) (Hemiptera: Tingidae) infesting cultivated olive trees in South Africa, and its complete mitochondrial sequence.

Olive lace bugs are small phytophagous Hemipteran insects known to cause agricultural losses in olive production in South Africa. Plerochila australis (Distant, 1904) has been reported as the species responsible for damage to olive trees; however, the diversity of olive lace bug species in the region has lacked attention. Adult olive lace bugs were collected incidentally from wild and cultivated olive trees in the Western Cape Province, and identified as P. australis and Neoplerochila paliatseasi (Rodrigues, 1981). The complete mitochondrial genome of a representative specimen of N. paliatseasi was sequenced, and used for comparative mitogenomics and phylogenetic reconstruction within the family. Furthermore, the value of DNA barcodes for species identification in Tingidae was assessed using genetic clustering and estimates of genetic divergence. The patterns of genetic clustering and genetic divergence of COI sequences supported the morphological identification of N. paliatseasi, and the utility of DNA barcoding methods in Tingidae. The complete mitogenome sequence had the typical Metazoan gene content and order, including 13 PCGs, 22 tRNAs, two rRNAs, and an AT-rich non-coding region. A+T content was high, as commonly found in Tingidae. The phylogenetic reconstruction recovered Agramma hupehanum (Drake Maa 1954) as basal to Tingini, and as a sister species to N. paliatseasi. Stephanitis Stål 1873 and Corythucha Stål 1873 were monophyletic, but Metasalis populi (Takeya 1932) was not recovered as sister to Tingis cardui (Linnaeus 1746), as expected. The mitochondrial phylogeny of the family Tingidae has been recovered inconsistently across different studies, possibly due to sequence heterogeneity and high mutation rates. Species diversity of olive lace bugs in South Africa was previously underestimated. The presence of P. australis was confirmed in both wild and cultivated olives, and N. paliatseasi is reported in cultivated olives for the first time. These results warrant further investigation on the diversity and distribution of olive lace bugs in the Western Cape to inform pest control strategies.