Lactic acid bacteria are recognized as food safety grade microorganisms (Saad et al., 2013). They contribute to improve food nutrition and ameliorate food flavor. At the same time, they have various probiotic functions such as the regulation of intestinal flora (Corinna et al., 2016) as well as the improvement of immunity (Akoglu et al., 2015). L. casei group, a type of lactic acid bacteria, can transit strong acid environments in the stomach and be colonized in the intestinal mucosa, thus play a major role in the prevention and treatment of gastrointestinal diseases. L. casei group have been widely commercialized. Several related products occupy a huge market share, and they are favored and affirmed by consumers. They have also expanded from the initial field of conventional yogurt to health, medicine, vaccines and several other fields. However, phage contamination is still a very serious problem for the industry of lactic acid bacteria (Garneau and Moineau, 2011). Phages can lyse bacteria to influence their death, decrease viable counts, cause slow fermentation and even production failure. These detriments consequently result in the decline of their acid production, flavor and taste. Since phages can resist pasteurization, their occurrence is difficult to completely eliminate. They are capable of rapid spread and even destruction of an entire production chain, leading to huge economic losses. Thus, anti-phage ability of lactic acid bacteria is a potential problem that needs to be explored in order to solve the problem of their development for useful applications.

Bacteria had evolved a variety of strategies against phages, which includes CRISPR/Cas system (Rodolphe et al., 2007; Garneau et al., 2010). These strategies prevent infection by cutting and integrating genetic elements of foreign invaders. Bacteria are also immune to other foreign invaders with homologous sequences (John et al., 2009; Makarova et al., 2011; Stern et al., 2012). CRISPR/Cas systems are usually clustered together with short palindromic repeats (CRISPR) and CRISPR-related (Cas) genes (Alexander et al., 2005; Rodolphe et al., 2007). CRISPR arrays contain RNA coding sequences that target foreign elements. Cas proteins function as nuclease and helicase, with the ability to unravel and cut DNA double strands in order to cause double-strand breaks in certain cases (Poorna et al., 2007; Tautvydas et al., 2013). In the past decade, CRISPR systems had been discovered to include 2 classes, 6 types, and more than 20 subtypes (Makarova et al., 2017a, b). Subsequently, researchers had developed them as gene-editing tools (Martin et al., 2012; Burgess, 2013; Wenyan et al., 2013). Currently, CRISPR-Cas9 gene-editing technology is widely used in animals and plants. CRISPR/Cas systems also have enormous potential applications as additions in genetic editing. Recently, the approach of strain-typing based on CRISPR system had been widely used in microorganisms such as Pseudomonas aeruginosa (Alex et al., 2015), salmonella enterica (Pettengill et al., 2014), and Helicobacter cinaedi (Tomida et al., 2017). Among lactic acid bacteria, it is usually difficult to type closely related strains based on the 16s rRNA sequences. However, this problem could be solved using CRISPR genotyping. Cas proteins capture fragments from foreign genetic elements and insert them into CRISPR array after processing to create a new repeat- spacer unit. The events of these insertions usually occur at the leader end of CRISPR loci, and the earlier acquired sequence, which also called ancestral spacers, is at the other end. As the evolution progresses, the newly acquired spacers are arranged in turn, then the information of the spacers’ position can form an evolution timeline. Bacteria rarely share exactly same CRISPR system. Thus, more useful background information is provided with CRISPR genotyping than other methods of genotyping.

Bioinformatic analysis of CRISPR system in probiotics is crucial for the assessment of potential evolution in the prediction of immunity, it is also of great importance to food industry and other applications. Our study would contribute to providing useful information about the molecular mechanism of L. casei group against phage infection. In addition, it would lay a foundation for subsequent screening and breeding of commercial anti-phage-infected Lactobacillus.

CRISPR system could resist foreign phages and plasmids through the mechanism of target interference by specific protein and guide RNA, thereby endue the bacteria strong adaptability to fight against complex environments (Makarova et al., 2013). Studies have found that about 40% of bacterial genomes contained CRISPR locus (Lillestøl et al., 2006), most CRISPR loci were located on their chromosomes and rarely on plasmids. The main reason for this phenomenon is that it will damage bacterial immunity hereditary if CRISPR-containing plasmids were lost. In the present study, we analyzed 68 strains of L. casei group, among which an extensive diversity was shown by CRISPR/Cas systems. Their different subtypes were harbored and included subtype IC, IE and IIA. It was obvious among different species there were diverse characteristics of CRISPR loci, thus provided a novel method of bacterial genotyping.

In general, direct repeat sequences of different CRISPR loci may exist differences. But they were conserved in same subtypes. Due to the presence of direct repeats of short palindromic sequences, double-stranded RNA secondary structures can be transcripted from the CRISPR array and combine with Cas proteins to target sites (Alexander et al., 2005; Tautvydas et al., 2013). The stem-loop structure of direct repeats may contribute to the interaction between RNA and Cas protein. As a consequence, the function of CRISPR loci may be affected by the stability of RNA secondary structures. Interestingly, in our study, the secondary structures of same subtypes were conserved, with similarities in their structural composition and free energy, irrespective of their differences in repeat sequences. It suggested that the repeat sequences were diverse in the process of evolution, although the function could be conserved. In addition, according to MFE value theory, longer match base numbers and higher GC contents in stem tended to form stable secondary structures, and secondary structures of RNA with lower minimum free energies were more stable. In understanding the evolution of bacteria, the genotyping analysis was crucial although excessive data analysis and high cost of sequencing had mainly hindered its widespread use. Another genotyping method commonly used is multilocus sequence typing (MLST), which is based on the nucleotide sequences of seven housekeeping genes. Tomida et al. (2017) determined a genotyping method based on CRISPR spacer and compared it with the methods of MLST genotyping using 42 H. cinaedi strains, the results showed MLST had little variability while the CRISPR spacer sequences showed remarkable diversity (Tomida et al., 2017). Morovic et al. (2016) showed that 42% of the commercial dietary supplements contained incorrectly labeled microorganism regarding taxonomy. Lewis et al. (2016) showed that 15 of 16 commercial probiotics in this study products present bacterial compositions that differed from the list of ingredients. Thus, the accessorial genotyping and correct identification methods were seriously needed as additions to traditional tools. The CRISPR/Cas systems had been used for the identification of various pathogens. However, rare reports are available about the application of genotyping to probiotics via CRISPR systems. Riedel et al. analyzed CRISPR systems and genotyped strains via spacer sequence in Bifidobacterium (Riedel et al., 2015; Hidalgo-Cantabrana et al., 2017), thereby created an awareness about the potential of the CRISPR system in probiotic genotyping. In this study, we considered CRISPR spacers as genotyping tools in L. casei group, in order to distinguish closely related strains. Different strains were specifically distinct, the later acquired spacers were diversified despite that they shared the same ancestral spacer. Only a few strains shared exact same spacers. Similar to other methods, the CRISPR/Cas genotyping method was largely limited and could be attributed to the absence of CRISPR systems in some strains. Therefore, the combination of multiple genotyping methods could be a developmental trend in the future. CRISPR spacers represent the immunity records of strains suffering from invasive DNA. The results of our study showed that less than half of spacers could match phages or plasmids. Only CECT9104 reached half of the spacers, while most of the other strains had only one or none. The limited number of spacer matches could be attributed to the presence of substantial plasmids or phages that had not been sequenced, or constantly evolve lead to escape mechanism. Strains obtained an evolutionary advantage from CRISPR-Cas systems by recording immune information, thereby prevented DNA invasion again. The L. casei group harboring CRISPR/Cas immune systems would be suitable as industrial probiotics against viral challenges. They could also have the potential to fight abundant phages in the gut.

In conclusion, our findings confirmed that L. casei group strains harbored diverse CRISPR/Cas systems. Furthermore, the results of the bioinformatic analysis could provide a data basis for broader CRISPR studies in L. casei group. The polymorphism of CRISPR system showed its potential for the genotyping of strains as well as the immunity of strains against invasive DNA. The CRISPR/Cas system analysis of L. casei group provided new insights into the diverse roles of CRISPR/Cas system in probiotic in this study.

The datasets generated for this study can be found in the https://www.ncbi.nlm.nih.gov/genome/genomes/652, https://www.ncbi.nlm.nih.gov/genome/genomes/2032.

LY, WL, YY, and XP collected the data and performed experiments. JL, SZ, XP, and JPL designed the study. LY and WL wrote the original draft. OU made multiple revisions and edited the manuscript. All authors contributed to the writing, reviewing, and editing of the manuscript. All authors read and approved the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.