Berges, C. | Search bioRxiv

bioRxiv2025

ZIP editors for efficient and versatile CRISPR-Cas9 precise genome editing

Precise genome editing technologies create the potential for genetic studies and innovative gene therapies. Here we present new CRISPR-Cas9 tools, named ZIP CRISPR, loaded with a single-stranded oligodeoxynucleotide (ssODN) template on the Cas ribonucleoprotein complex. The ssODN template is annealed to an extended guide RNA (gRNA) allowing its nuclear delivery at the right place, i.e. the targeted DNA cut, and at the right time. This new template import system is easy-to-design, easy-to-use, inexpensive and versatile. It increases homology-directed repair (HDR) editing efficiency using Cas9 nuclease up to 12-fold with a mean increase of 5-fold as demonstrated at many loci in many cell types. Based on a heteroduplex gRNA-ssODN, it can also be used with the Cas9 nickase, resulting in HDR editing with minimal InDels, and preventing double-strand break (DSB)-mediated genotoxicity. ZIP CRISPR is a non-viral platform adaptable to targeted DSB (higher HDR editing efficiency) or nick (higher safety) to precisely model and correct a wide range of edits. It is suitable for many biological applications and could be considered for HDR-based gene therapies.

bioengineering↗

bioRxiv2022

Validation of carbon isotopologue distribution measurements by GC-MS and application to 13C-metabolic flux analysis of plant tricarboxylic acid cycle

The estimation of metabolic fluxes in photosynthetic organisms represents an important challenge that has gained interest over the last decade with the development of 13C-Metabolic Flux Analysis at isotopically non-stationary steady-state. This approach requires a high level of accuracy for the measurement of Carbon Isotopologue Distribution in plant metabolites. But this accuracy has still not been evaluated at the isotopologue level for GC-MS, leading to uncertainties for the metabolic fluxes calculated based on these fragments. Here, we developed a workflow to validate the measurements of CIDs from plant metabolites with GC-MS by producing tailor-made E. coli standard extracts harboring a predictable binomial CID for some organic and amino acids. Overall, most of our TMS-derivatives mass fragments were validated with these standards and at natural isotope abundance in plant matrices. Then, we applied this validated MS method to investigate the light/dark regulation of plant TCA cycle by incorporating U-13C-pyruvate to Brassica napus leaf discs. We took advantage of pathway-specific isotopologues/isotopomers observed between two and six hours of labeling to show that the TCA cycle can operate in a cyclic manner under both light and dark conditions. Interestingly, this forward cyclic flux mode has a nearly four-fold higher contribution for pyruvate-to-citrate and pyruvate-to-malate fluxes than the phosphoenolpyruvate carboxylase (PEPc) flux reassimilating carbon derived from some mitochondrial enzymes. The contribution of stored citrate to the mitochondrial TCA cycle activity was also questioned based on dynamics of 13C-enrichment in citrate, glutamate and succinate and variations of citrate total amounts under light and dark conditions. Interestingly, there was a light-dependent 13C-incorporation into glycine and serine showing that decarboxylations from pyruvate dehydrogenase complex and TCA cycle enzymes were actively reassimilated and could represent up to 5% to net photosynthesis.

plant biology↗