Dataset Information

Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from Ruminococcus bromii.

ABSTRACT: Type V Cas12a nucleases are DNA editors working in a wide temperature range and using expanded protospacer-adjacent motifs (PAMs). Though they are widely used, there is still a demand for discovering new ones. Here, we demonstrate a novel ortholog from Ruminococcus bromii sp. entitled RbCas12a, which is able to efficiently cleave target DNA templates, using the particularly high accessibility of PAM 5'-YYN and a relatively wide temperature range from 20 °C to 42 °C. In comparison to Acidaminococcus sp. (AsCas12a) nuclease, RbCas12a is capable of processing DNA more efficiently, and can be active upon being charged by spacer-only RNA at lower concentrations in vitro. We show that the human-optimized RbCas12a nuclease is also active in mammalian cells, and can be applied for efficient deletion incorporation into the human genome. Given the advantageous properties of RbCas12a, this enzyme shows potential for clinical and biotechnological applications within the field of genome editing.

SUBMITTER: Vasilev R

PROVIDER: S-EPMC9409102 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from Ruminococcus bromii.

Vasilev Ruslan R Gunitseva Natalia N Shebanova Regina R Korzhenkov Aleksei A Vlaskina Anna A Evteeva Marta M Polushkina Irina I Nikitchina Natalia N Toshchakov Stepan S Kamenski Piotr P Patrushev Maxim M Mazunin Ilya I

International journal of molecular sciences 20220818 16

Type V Cas12a nucleases are DNA editors working in a wide temperature range and using expanded protospacer-adjacent motifs (PAMs). Though they are widely used, there is still a demand for discovering new ones. Here, we demonstrate a novel ortholog from Ruminococcus bromii sp. entitled RbCas12a, which is able to efficiently cleave target DNA templates, using the particularly high accessibility of PAM 5'-YYN and a relatively wide temperature range from 20 °C to 42 °C. In comparison to Ac ...[more]

PMID: 36012553

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Project description:Dietary and host glycans shape the composition of the human gut microbiota with keystone carbohydrate-degrading species playing a critical role in maintaining the structure and function of gut microbial communities. Here, we focused on two major human gut symbionts, the mucin-degrader Ruminococcus gnavus ATCC 29149, and R. bromii L2-63, a keystone species for the degradation of resistant starch (RS) in human colon. Using anaerobic individual and co-cultures of R. bromii and R. gnavus grown on mucin or starch as sole carbon source, we showed that starch degradation by R. bromii supported the growth of R. gnavus whereas R. bromii did not benefit from mucin degradation by R. gnavus. Further we analyzed the growth (quantitative PCR), metabolite production (1H NMR analysis), and bacterial transcriptional response (RNA-Seq) of R. bromii cultured with RS or soluble starch (SS) in the presence or absence of R. gnavus. In co-culture fermentations on starch, 1H NMR analysis showed that R. gnavus benefits from transient glucose and malto-oligosaccharides released by R. bromii upon starch degradation, producing acetate, formate, and lactate as main fermentation end-products. Differential expression analysis (DESeq 2) on starch (SS and RS) showed that the presence of R. bromii induced changes in R. gnavus transcriptional response of genes encoding several maltose transporters and enzymes involved in its metabolism such as maltose phosphorylase, in line with the ability of R. gnavus to utilize R. bromii starch degradation products. In the RS co-culture, R. bromii showed a significant increase in the induction of tryptophan (Trp) biosynthesis genes and a decrease of vitamin B12 (VitB12)-dependent methionine biosynthesis as compared to the mono-culture, suggesting that Trp and VitB12 availability become limited in the presence of R. gnavus. Together this study showed a direct competition between R. bromii and R. gnavus on RS, suggesting that in vivo, the R. gnavus population inhabiting the mucus niche may be modulated by the supply of non-digestible carbohydrates reaching the colon such as RS.

Dataset Information

Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from Ruminococcus bromii.

Publications

Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from <i>Ruminococcus bromii</i>.

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