William Rostain's research while affiliated with Institut Pasteur and other places

... In comparison, Cas9 requires a PAM and at least five nucleotides of seed complementarity to stably associate with non-target sequences. This stable association of Cas9 with non-target sequences has been demonstrated to cause cellular toxicity (28,34). These observations emphasize the importance of carefully considering the implications of SpRY and Cas9 behavior to mitigate off-target effects and enhance the safety of gene editing applications. ...

... The gRNA (guide RNA) is a small synthetic sequence of RNA that functions as a guide for DNA targeting enzymes. These enzymes are used for various purposes like deletion, insertion, or targeting of RNA (Collins et al., 2021). The design of gRNA can be automated through the use of various tools available online. ...

... Recent studies have demonstrated that CRISPR activity could be controlled by sensing endogenous RNA molecules. [13][14][15][16][17][18][19][20][21][22][23][24] This was achieved by either engineering expression cassettes encoding CRISPR components or by designing RNA-responsive sgRNAs. An example of an engineered expression cassette involves miRNA sensing by introducing miRNA-responsive elements (MREs) within Cas9, sgRNA, or anti-CRISPR transcripts. ...

... Ford and Ares (1994) replicated this strategy by permuting the intron from the thymidylate synthase (td) gene of T4 bacteriophage to circularize the td exon [66]. Since then, the permuted T4 td and Anabaena pre-tRNA genes have become staple backbones in the field for circularization of a wide assortment of exonic sequences inserted between the group I intron halves [67][68][69][70][71][72][73][74][75][76]. Wesselhoeft et al. (2018) reported significant increases in circRNA yield when a luciferase sequence was transferred from a PIE construct with td introns to one containing Anabaena pre-tRNA introns [75], suggesting that choice of group I intron affects circularization efficiency. ...

... The cre/lox system has been used as a deletion system on many occasions, due to its ability to act in both prokaryotic and eukaryotic cells. In addition, the usage of mutant lox sites to facilitate deletions that result in an inactive lox has also been demonstrated in bacteria, such as being used to knock out single genes in series (Pan et al., 2011), or to knock out large but targeted genome region using either targetrons (Cerisy et al., 2019) or via recombineering (Xin et al., 2018). ...

... These results indicate PTS-dependent processing of the 12 molecules. Although we cannot exclude that other functional elements could be involved in the transport of the substrates tested herein (e.g., hexose permeases or facilitators, which could promote growth on glucose or fructose) [18][19][20] . In addition to the 12 carbon substrates on which growth was abolished, N-acetylmuramic acid yielded diminished growth indicating that this substrate is mainly, but not exclusively, metabolized through PTS. ...

... Ther efor e, utilization of RNA molecules to construct synthetic circuits for cell engineering has a great advantage of applicable generality and designable flexibility, allowing expression of different types of genes to be modulated at the RNA le v el regar dless of what functions these genes are related to. Taking advantage of this unique feature of RNA-based circuits, many efforts have shown that biomolecules, including RNAs, proteins and small molecules, can be programmed to manipulate gene expression through either transcriptional or translational control (3)(4)(5). These RNA circuits can work on the 2 Nucleic Acids Research, 2023 basis of different molecular machineries, including but not limited to , ribos witches (6)(7)(8), riboregulators (9)(10)(11)(12)(13), small interfering RNAs (siRNAs) (14)(15)(16) and clustered regularly interspaced palindromic repeats (CRISPR) technologies (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). ...

... The structure of the targeted GGA cassette ruled the composition of the GG reaction mixture. The Golden Gate reaction conditions were designed based on the previously published protocols (Engler et al., 2008;Pauthenier et al., 2012;Agmon et al., 2015). The reaction mixture contained precalculated equimolar amount of each GGF and the destination vector (50 pmoles of ends), 2 ll of T4 DNA ligase buffer (NEB), 5 U of BsaI, 200 U of T4 and ddH2O up to 20 ll. ...

... The simplest topological difference between 24mers is linear versus circular. Indeed, circular ssRNAs are known to have a slower electrophoretic mobility as compared to the linear molecules of equal length, 61 in line with the position of this extra band of different mobility. ...

... The thermodynamic properties of all designed sRNAs (rtRNAs and asRNA) were evaluated using the NUPACK Web Application (Zadeh et al., 2011). sRNAs showing ensemble defect higher than 10%, and the complex representation at equilibrium lower than 96% were discarded (Rostain et al., 2015). One rtRNA targeting each riboswitch and one asRNA targeting purR were selected for construction and test (Tables S3 and S4). ...