The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)‍-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.
CRISPR-Associated Protein 9/metabolism* ; CRISPR-Cas Systems ; DNA ; Gene Editing/methods* ; Humans ; Streptococcus pyogenes/metabolism*

Lysins are murein hydrolases produced by bacteriophage that act on the cell wall of host bacteria to release progeny phages. Research indicated that lysins could kill bacteria effectively and specifically in vitro. To prepare recombinant bacteriophage lysin of Streptococcus (PlyC) and analyze its biological activity, we obtained two genes of PlyC named PlyCA and PlyCB by PCR amplification and inserted them into pET-32a(+), then transformed the recombinant expression vectors pET-32a(+)-PlyCA and pET-32a(+)-PlyCB into E. coli BL21(DE3) respectively. After induction with 0.7 mmol/L IPTG at 30 degrees C for 7 h, PlyCA and PlyCB were successfully expressed, SDS-PAGE analysis determined that they all constituted above 30% of the total cell proteins. After Ni(2+)-NTA affinity chromatography, the purity was more than 95%. With the denaturation and protein refolding, we gained the recombinant PlyC. To determine its biological activity, we adopted turbidimetry and plate count method. Before and after lysin treatment, the cell morphology was studied by scanning electron microscopy (SEM). The results showed that the recombinant PlyC could specifically cleavage Streptococcus pyogenes (group A beta-hemolytic streptococci). Under the incubation time of 60 min with 4 microg/mL PlyC in Streptococcus pyogenes dilution which OD600 was 0.56, the germicidal effect was up to 99.6%, while SEM observations showed that cell wall cracked and presented cell debris. This finding laid the foundation for the further study and achieving an effective treatment for streptococcal infection.
Bacteriolysis ; Enzymes ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Streptococcus pyogenes ; drug effects

We amplified genes encoding UDP-glucose dehydrogenase, ecohasB from Escherichia coli and spyhasB from Streptococcus pyogenes. Both ecohasB and spyhasB were inserted into T7 expression vector pRX2 to construct recombinant plasmids pRXEB and pRXSB, and to express in E. coli BL21(DE3). After nickel column purification of UDP-glucose dehydrogenases, the enzymes were characterized. The optimum reaction condition of spyHasB was at 30 °C and pH 10. The specific activity reached 12.2 U/mg under optimum condition. The optimum reaction condition of ecoHasB was at 30 °C and pH 9. Its specific activity reached 5.55 U/mg under optimum condition. The pmuhasA gene encoding hyaluronic acid synthase was amplified from Pasteurella multocida and ligated with ecohasB and spyhasB to construct the coexpression vectors pBPAEB and pBPASB, respectively. The co-expression vectors were transformed into E. coli BW25113. Hyaluronic acid (HA) was produced by biotransformation and the conditions were optimized. When recombinant strains were used to produce hyaluronic acid, the higher the activity of UDP-glucose dehydrogenase was, the better its stability was, and the higher the HA production could reach. Under the optimal conditions, the yields of HA produced by pBPAEB/BW25113 and pBPASB/BW25113 in shake flasks were 1.52 and 1.70 g/L, respectively, and the production increased more than 2-3 folds as previously reported.
Biotransformation ; Escherichia coli ; enzymology ; Genetic Vectors ; Glucuronosyltransferase ; genetics ; Hyaluronan Synthases ; Hyaluronic Acid ; metabolism ; Pasteurella multocida ; enzymology ; Streptococcus pyogenes ; enzymology ; Uridine Diphosphate Glucose Dehydrogenase ; metabolism

Streptococcus pyogenes Cas9 (SpCas9) has become a powerful genome editing tool, but has a limited range of recognizable protospacer adjacent motifs (PAMs) and shows off-target effects. To address these issues, we present a rational approach to optimize the xCas9 mutant derived from SpCas9 by directed evolution. Firstly, energy minimization with the Rosetta program was applied to optimize the three-dimensional structure of Cas9 to obtain the lowest energy conformation. Subsequently, combinatorial mutations were designed based on the mutations sites of xCas9 acquired during the directed evolution. Finally, optimal mutants were selected from the designed mutants by free energy ranking and subjected to experimental verification. A new mutant yCas9 (262A/324R/409N/480K/543D/694L/1219T) with multiple PAM recognition ability and low off-target effects was obtained and verified by DNA cleavage experiments. This mutant recognizes the NG, GAA and GAT PAMs and shows low off-target DNA cleavage activity guided by mismatched sgRNA, thus provides a gene editing tool with potential applications in biomedical field. Furthermore, we performed molecular dynamics simulations on the structures of SpCas9, xCas9 and yCas9 to reveal the mechanisms of their PAM recognition and off-target effects. These may provide theoretical guidance for further optimization and modification of CRISPR/Cas9 proteins.
CRISPR-Associated Protein 9/metabolism* ; CRISPR-Cas Systems/genetics* ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; RNA, Guide/genetics* ; Streptococcus pyogenes/metabolism*