CRISPR-Cas9: Gene Scissors in the Microbial World

The rapid development of genome editing technology has enabled precise and efficient genome modification in the field of microorganisms. This technology not only enables scientists to better understand the biological characteristics of microorganisms, but also provides unprecedented possibilities for fields such as biopharmaceuticals, industrial production, and environmental protection. CRISPR-Cas9 is a revolutionary gene editing technology, and its widespread application is changing the field of biological science. In microbiology, the application of CRISPR-Cas9 technology provides researchers with a powerful tool to accurately edit microbial genomes, explore their functions, regulate metabolic pathways, and develop new biotechnology.

Currently, nuclease based genome editing technology has entered the third generation, including ZFN (zinc finger nuclease), TALEN (transcriptional activated genome editor), and CRISPR Cas system. These technologies rely on DNA repair systems induced by double strand breaks (DSB), which modify sequences at specific locations. However, ZFN technology generates cytotoxicity within cells and has high production costs. In contrast, TALEN has high fidelity and fewer off target effects, but its module assembly is more complex. Compared with the first two technologies, the CRISPR Cas system has a wider range of target selection, avoiding assembly difficulties and off target issues. In addition, the CRISPR Cas system can effectively cleave any DNA site, achieving more precise gene editing and modification.


The mechanism of CRISPR-Cas9 system

The CRISPR-Cas9 system consists of three basic components: the CRISPR locus, tracrRNA, and Cas9 endonuclease. The CRISPR locus consists of spacer and repeat sequences, with repeat sequences typically ranging from 21 to 48 bp in length and spacer sequences ranging from 26 to 72 bp, responsible for identifying exogenous DNA. The RNA double stranded body formed by tracrRNA and crRNA works together with Cas9 endonuclease to scan the PAM (adjacent motif of the prototype spacer) on the target DNA to guide double stranded cleavage. PAM is a special sequence located on the target DNA of the CRISPR site, typically 5 ‘upstream of the prototype spacer in the Type I system and 3’ downstream of the prototype spacer in the Type II system.
Cas9 endonuclease contains the RuvC domain located at the N-terminus and the HNH nuclease active domain in the middle, which can induce double stranded cleavage of specific DNA sequences. The discovery of this system can be traced back to 2007, when Barrangou et al. observed CRISPR Cas mediated adaptive immunity in Streptococcus thermophilus. They found that some strains of Streptococcus thermophilus survived after phage infection, and their CRISPR structures obtained new spacer regions that partially matched the genomic sequences of the phages used for infection. The newly obtained spacer zone endows these strains with resistance to corresponding bacteriophages.

The immune process of the CRISPR-Cas9 system is mainly divided into three steps: adaptation, expression, and interference. The adaptation stage is when bacteria are invaded by exogenous DNA, and the system integrates a portion of these DNA fragments into its own genome to construct new CRISPR spacer regions. During the expression stage, the sequence of the CRISPR spacer is transcribed and processed to form crRNA, which forms a double stranded structure with tracrRNA. Finally, during the interference phase, this double stranded RNA structure will bind to Cas9 endonuclease to recognize and cleave foreign DNA that matches it, in order to resist foreign invasion.

Figure 1. The three stages of CRISPR-Cas9 immune system and their applications.

Adaptation: The adaptation stage of the CRISPR structure involves selectively integrating exogenous DNA fragments as new spacer sequences into the CRISPR structure, thereby forming the process of immune memory. When exogenous DNA, such as viruses or plasmids, invades prokaryotic cells, these DNA are recognized and cleaved into specific fragments by Cas protein complexes (mainly Cas1 and Cas2), a process known as spacer selection. Usually, the interval is obtained through the specific recognition of PAM. Researchers have found that Cas9 may be responsible for identifying PAM and recruiting Cas1-Cas2 complexes in type II systems. Subsequently, these fragments are specifically integrated into specific sites of the CRISPR DNA structure (usually adjacent to the lead sequence), while the repetitive sequences of the integration sites are precisely replicated. Therefore, the periodicity of the CRISPR structure can be maintained, and this sub stage can be referred to as the “interval integration process”.

Expression: The newly obtained spacer region needs to be transcribed and processed into mature small molecule crRNA to mediate specific immunity. This process constitutes the second stage of CRISPR immunity, which is the biosynthesis of crRNA. Most Type II systems encode tracrRNAs that partially complement repetitive sequences. Usually, the leading sequence of CRISPR structure contains a promoter and other transcription elements. When the same virus or plasmid invades the bacteria again, the inserted new interval sequence will be transcribed together with the repeated sequence. Under the guidance of tracrRNA, host derived RNase III and Cas9 cleave duplicate RNA, and then perform a second cleavage near the 5 ‘end of the spacer sequence. Ultimately, the length of mature crRNA molecules ranges from 44 to 49 nt, with the 5 ‘end of the 20-22 nt derived from the spacer sequence and the 3’ end of the 24-27 nt derived from the repeat sequence.
Interference: Functional crRNA guides Cas9 nuclease to specifically recognize and cleave viral DNA/RNA homologous to the spacer region, thereby achieving specific immune processes. After RNase III treatment, mature crRNA and tracrRNA continue to form RNA double stranded bodies. RNA double stranded and Cas9 will quickly scan the PAM sequence on the target DNA. The base matching between crRNA and the DNA of the prototype spacer region is extended to the entire prototype spacer region, forming a stable R-ring structure. The HNH domain and Ruv-C domain cleave two strands of the target DNA, resulting in double strand breakage.

CRISPR-Cas9 bacterial genome editing strategy

The bacterial genome editing method based on CRISPR-Cas9 has rapidly developed since its discovery and has become one of the main genome editing tools in many organisms. At present, various editing methods based on CRISPR-Cas9 have been developed, and their differences mainly lie in the number of plasmids used, whether heterologous recombinases (such as lambda Red) are used, and the induced DNA repair mechanism (such as HDR).

 CRISPR Cas based genome editing strategy in bacteria

Figure 2. Genome editing strategy based on CRISPR Cas in bacteria.

The most commonly used strategy in Escherichia coli and other model bacteria is to use DNA linear templates and recombinant enzymes derived from bacteriophages to repair DSB (double stranded breaks). In this method (see Figure 2A), the expression of exogenous recombinase is first introduced into the plasmid, and then transformed together with the recombinant DNA template and CRISPR plasmid (Cas9+gRNA). However, the bottleneck of this method lies in the availability of efficient recombinant enzymes and the selection of a sufficient number of feasible gene editing colonies from unedited colonies. The initial description of genome editing in Escherichia coli used SpCas9 and lambda Red bacteriophage proteomic enzyme systems and linear double stranded DNA templates to achieve the required editing. This system has been successfully applied to other species, mainly Proteobacteria. Recently, the activity of other heterologous recombination systems has also been screened for different bacteria, whether they are used alone or combined with CRISPR Cas.

Another method is that DNA repair templates can be encoded in plasmids that are the same or different from SpCas9. In this case, exogenous recombinant enzymes have been used, although natural recombination mechanisms can also be relied upon. In some works, it has been confirmed that this method can be successfully achieved using recombinant template plasmids in Escherichia coli with a 1Kb homologous arm. Vento et al.’s study described other bacteria, among which natural recombination mechanisms have been successfully applied, such as Clostridium ljungdahlii, Lactobacillus plantarum, Pseudomonas putida, Streptomyces azureus, and Staphylococcus aureus. Although natural recombination mechanisms can simplify systems, in many species, these mechanisms are either unreliable or inefficient enough to achieve the required editing.

When repairing CRISPR Cas induced double stranded breaks through non homologous end effector pathways, the recombination template and/or mechanism can also be omitted (see Figure 2B). However, few bacterial species have sufficiently active NHEJ mechanisms themselves, so heterologous coding in CRISPR plasmids is usually required. The NHEJ mechanism in bacteria is mainly composed of two proteins: Ku and LigD. Ku binds to the cleaved ends of DNA, while LigD connects them, often introducing non-specific mutations, insertions, or deletions, leading to loss of gene function. Similarly, the natural alternative terminal junction (A-EJ) pathway, also known as microhomology mediated junctions, can be utilized. This DNA repair pathway relies on microhomology near the Cas9 cleavage site. After the end of the DNA is cleaved by RecBCD, Cas9 is connected by LigA, leaving a variable size deletion after repair. Natural A-EJ has been combined with CRISPR-Cas9 for application in multiple species, including Escherichia coli, Streptomyces aeruginosa, and Fructobacter niger. Neither of these strategies can introduce specific mutations or insertions, but they are effective for gene knockout.

Overall, these strategies are not mutually exclusive and can be combined based on the characteristics of the host species. However, it should be noted that they may share some drawbacks related to the continuous expression of exogenous Cas9 protein. Overexpression of SpCas9 in Escherichia coli and many other bacteria may have high cytotoxicity, even without its nuclease activity, it may lead to little or no colony formation.

Application of CRISPR-Cas9 in other microorganisms

Pichia pastoris genome editing

Pichia pastoris is a common yeast widely used for fermenting food and brewing alcoholic beverages. Using CRISPR-Cas9 technology, researchers can quickly and accurately edit the genome of Pichia pastoris to improve its fermentation performance, increase yield, and enhance its stress resistance. For example, by editing genes in key metabolic pathways, Pichia pastoris can increase its utilization efficiency of specific waste or inexpensive substrates, thereby improving the production efficiency of alcohol or biodiesel.

Genome editing of Bacillus subtilis

Bacillus subtilis is a widely present Gram positive bacterium in soil, with important industrial and biological application value. By utilizing CRISPR-Cas9 technology, researchers can achieve precise editing of the genome of Bacillus subtilis to improve its ability to produce enzymes, trypsin, and other industrial enzymes. In addition, genome editing can enhance the stress resistance of Bacillus subtilis and improve its application in the production of biological insecticides and fertilizers.

Genome editing of brewing yeast

Brewing yeast is an important microorganism for brewing beer and fermenting alcoholic beverages. Using CRISPR-Cas9 technology, researchers can edit the genome of brewing yeast to improve its fermentation performance, adjust beer taste, and increase yield. For example, by knocking out specific metabolic pathways or regulating the expression levels of key genes, the utilization efficiency of brewing yeast for different fermentation substrates can be adjusted, thereby improving the quality and taste of beer.

Lactic acid bacteria genome editing

Lactic acid bacteria are an important class of Gram positive bacteria, widely used in food processing and lactic acid fermentation industry. Using CRISPR-Cas9 technology, researchers can perform precise genome editing on lactic acid bacteria to improve their fermentation performance, acid production efficiency, and stress resistance. By editing genes in key metabolic pathways, the utilization efficiency of lactic acid bacteria in different food ingredients can be enhanced, thereby improving the quality and nutrition of lactic acid fermented foods.

Salmonella genome editing

Salmonella is a type of pathogenic bacteria that can cause diseases such as food poisoning and intestinal infections. By utilizing CRISPR-Cas9 technology, researchers can achieve precise genome editing of Salmonella to study its pathogenic mechanisms, develop new treatment methods, and improve food safety. By knocking out or targeting the virulence factor genes of Salmonella, its pathogenicity can be reduced, thereby reducing the occurrence of food poisoning events.

Genome editing of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram negative bacterium widely present in soil and water, and is also a common pathogenic bacterium that can cause hospital infections and cystic fibrosis. Using CRISPR-Cas9 technology, researchers can edit the genome of Pseudomonas aeruginosa to study its pathogenic mechanism, develop new antibiotics, and biological control methods. Targeted editing of resistance and virulence factor genes of Pseudomonas aeruginosa can reduce its resistance to antibiotics and improve treatment efficacy.

CRISPR/Cas9 technology is an important step in genetic engineering, with a wide and far-reaching range of applications. CRISPR-Cas9, as a new generation of gene editing technology, has broad application prospects in gene function research, model animal construction, disease gene therapy, and other fields. Although CRISPR/Cas9 technology has brought profound impact and enormous potential, it also faces certain challenges and problems. For example, it may lead to non-specific gene editing, causing unexpected mutations. In addition, there are ethical issues, including human embryonic gene editing and gene enhancement. In summary, CRISPR/Cas9 is a powerful biological tool with extensive applications. Despite the challenges, with the progress of scientific research and the efforts of researchers, we have sufficient reason to believe that CRISPR/Cas9 can bring broader prospects to human society.