CRISPR-B (Clustered Regularly Interspaced Short Palindromic Repeats-B) gene editing method for gtfB site of specific targeting streptococcus mutans and application

Abstract

The invention discloses a CRISPR-B (Clustered Regularly Interspaced Short Palindromic Repeats-B) gene editing method for a gtfB site of specific targeting streptococcus mutans and application. The gene sequence is shown as SEQ ID NO. 1; the gene editing method comprises the following steps: step 1: connecting a lactic dehydrogenase promoter Ldhp in front of the gene sequence to form Ldhp+CRISPR-B;step 2: connecting the Ldhp+CRISPR-B to a linear plasmid pDL278 through enzyme digestion and connection, so as to form a CRISPR editing vector; step 3: taking an upstream sequence of a target gene gtfB, knocked out by polymerase chain reaction amplification, as upstream; step 4, taking a downstream sequence of a target gene gtfC, knocked out by the polymerase chain reaction amplification, as downstream; step 5: connecting the upstream obtained step 3 and the downstream obtained by step 4 to form a homologous recombination template HR-BC; step 6: converting the CRISPR editing vector obtained by step 2 and the homologous recombination template HR-BC obtained by step 5 into the streptococcus mutans, so as to simultaneously edit the gtfB and the gtfC. By adopting the CRISPR-B gene editing method disclosed by the invention, the synthesis of streptococcus mutans extracellular polysaccharides and the formation of a biological film can be inhibited to the greatest extent.

Description

technical field [0001] The invention relates to gene sequences, in particular to a CRISPR-B gene editing method and application specifically targeting the gtfB site of Streptococcus mutans. Background technique [0002] Caries is one of the common oral diseases in humans, and it is a chronic infectious disease caused by cariogenic microorganisms metabolizing acid production (Petersen P E, Kwan S. World Health Organization global oral health strategies for oral health promotion and disease prevention in the twenty- first century[J].Prvention Und Gesundheitsfrderung, 2009,4(2):100-104.); Streptococcus mutans (Streptococcus mutans) is one of the world-recognized cariogenic microorganisms, and its cariogenic characteristics are mainly reflected in the following aspects Aspects: strong acid production and acid resistance, strong environmental adaptability, synthesis of extracellular insoluble polysaccharides (Extracellular polysaccharide, EPS) and strong adhesion to tooth surface...

AI Technical Summary

Problems solved by technology

[0004] Up to now, most of the methods for inhibiting the Gtfs activity of Streptococcus mutans are by adding small molecules such as some natural organic compounds or inorganic compounds in vitro, such as: flavonoids, quinoxaline imines and fluorides, etc. (Ren Z, Chen L, LiJ, et al.Inhibition of Streptococcus mutans polysaccharide synthesis bymolecules targeting glycosyltransferase activity[J].Journal of Oral Microbiology,2016,8(1):31095.); Although they can well inhibit the activity of Gtfs, there are still some deficiencies : First, due to the blindness of the screening, small molecules with strong specificity cannot be found efficiently and accurately; second, the specific mechanism of action is mostly unclear and has not been systematically elucidated; third, although some small molecules have strong It is not yet known whether there are any specific side effects; fourth, the purification or synthesis of some small molecules is expensive and the steps are cumbersome; fifth, although some compounds come from natural medicines, it is not known whether they will bury potential toxicity. report; sixth, whether the physical and chemical properties of small molecules (such as solubility, action time, etc.) are conducive to inhibiting Gtfs activity, etc.; in addition, there are also traditional gene knockout methods to knock out gtfB, gtfC or gtfD, although there are It can achieve the inhibitory effect, but there are disadvantages such as low efficiency, cumbersome steps and poor specificity; therefore, based on the above reasons, it is urgent to find a new strategy to make up for the shortcomings of the current technology

The type II-A CRISPR / Cas9 system locus structure includes: 5'-end tracrRNA (trans-activating CRISPRRNA) gene, which is mainly combined with mature crRNA to form a complex to guide Cas9 protein to specifically cut foreign DNA; the middle is a A series of Cas protein coding genes (including Cas9, Cas1, Cas2 and Csn2), which mainly play the role of nuclease cleavage; and the CRISPR locus at the 3' end: composed of promoter regions, a large number of spacers and repeat sequences (directrepeats) sequence The spacers are DNA from viruses or plasmids; in addition, the main function of the protospacer adjacent motif (PAM: NGG) is to help Cas9 to accurately distinguish non-self DNA that needs to be degraded The sequence is identical to its own DNA, so as to achieve specific targeted cutting (Jinek M, Chylinski K, Fonfara I, et al.A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity[J]. ):816.); Therefore, when bacteria encounter the invasion of foreign DNA again, the CRISPR locus will transcribe and produce tracrRNA and crRNA, and combine with Cas9 to form a minimal functional complex, and then crRNA will specifically guide Cas9 to target clipping exogenous DNA; however, this technique is still in its infancy in the study of caries microbes, especially gene editing of Streptococcus mutans

Images