The features of clustered regularly interspaced short palindromic repeats (CRISPR) were discovered serendipitously in the genomes of various bacteria and archaea during molecular biology studies (Ishino et al., 1987; Mojica et al., 1993). The system was originally used as an adaptive immune defense system by bacteria against the invasion of foreign nucleic acids, such as viruses and plasmids (Ishino et al., 1987; Sovova et al., 2017; Albitar et al., 2018). The immune process is divided into three stages: 1) adaptation: the invading nucleotide fragments are first captured by the host organism, and subsequently, a Cas integrase-derived nucleic acid sequence is inserted into the CRISPR array; 2) biogenesis: the CRISPR array is transcribed into a pre-crRNA (pre-crRNA) containing a spacer and a portion of the repeat, which is subsequently cleaved in the repeat to induce the generation of mature guide crRNA (gRNA); and 3) interference: mature crRNA-Cas complexes recognize specific sites on target nucleic acids through complementary base pairing, triggering Cas enzymes to catalyze the cleavage of effector complexes of invading nucleotides (Debin Zhang et al., 2020; Makarova et al., 2020). CRISPR provides acquired immunity to prokaryotes, in addition to other unrelated functions, such as gene regulation (Mojica and Rodriguez-Valera, 2016). The discovery and application of the CRISPR-Cas enzyme system in prokaryotes and eukaryotes have revolutionized genome engineering by providing directly accessible or editable tools (Barrangou, 2014).

CRISPR/Cas systems usually consist of Cas proteins and CRISPR arrays, in which the Cas proteins are involved in the acquisition and protection of invading nucleotides. Currently, CRISPR/Cas systems are divided into classes 1 and 2 according to the composition of their effectors. Class 1 systems include many Cas protein subunits, including types I, III, and IV (Makarova et al., 2011). In contrast, only one Cas protein involving II, V, and VI subdomains is included in Class 2 (Ishino et al., 2018). Class 2 CRISPR/Cas systems have emerged as an attractive option for developing next-generation genome-editing technologies because of their simple structural design and easy implementation in gene editing and manipulation of cell-free nucleic acids (Tang and Fu, 2018; Debin Zhang et al., 2020). Therefore, we consider a typical Class 2 system as an example to introduce the basic components of the system.

Generally, the CRISPR/Cas9 system is considered the smallest CRISPR/Cas system because its pre-CrRNA processing occurs solely with Cas9, a member of the Cas family. Apart from Cas9, single-guide RNA (sgRNA) is the other main component of this simple and easy Class 2 system. sgRNA structure comprises two parts, one of which plays a role in guiding cleavage by binding to Cas9 (Mojica et al., 2005; Pourcel et al., 2005), whereas the other is capable of binding to the target sequence through complementary base pairing to guide positioning. A conserved sequence called the proto-spacer adjacent motif (PAM) exists downstream of the target sequence (Jinek et al., 2012). The presence or absence of the PAM sequence determines whether Cas9 protein cleaves the target sequence. In this system, Cas9 cleaves the target DNA sequence under the guidance of gRNA to create double-strand breaks (DSB). CRISPR systems in prokaryotes often require the addition of donor DNA fragments because they only have homology-directed repair (HDR) (Liang et al., 1998). In addition, the deletion or insertion of target DNA sequences can also be achieved by the addition of donor DNA fragments. Eukaryotes have access to another mode of non-homologous end joining (NHEJ) repair, allowing bacteria to repair without DSBs in the presence of donor DNA (Lieber, 2010). Another vital element, the Cas9 protein, is regarded as a nuclease with two cleavage domains: RuvC and HNH (Deltcheva et al., 2011). While both domains are activated, the HNH nuclease domain cleaves the DNA strand complementary to the sgRNA, and RuvC cleaves the DNA strand that is not complementary to the sgRNA, forming a nicked double-strand break near the PAM. Based on these properties, Cas9 can be directed to the target gene to generate DSBs by designing the sgRNA spacer sequence. In some studies, the two nuclease domains of RuvC and HNH were mutated to introduce novel Cas9 or completely inactivate Cas9, which has excellent effects in improving target efficiency (Makarova et al., 2011).

In recent years, the emergence of CRISPR/Cas and its derivative editing technology has become a pillar of experimental biology, as well as a tool for synthetic biology, because of convenient implementation, high mutation efficiency, and great potential for therapeutic applications (Zeballos and Gaj, 2021). However, the system itself has the risk of being off-target, with low multiple editing efficiency. The toxicity of Cas9 affects the broad application of this gene-editing technology in cell factories and synthetic biology (Figure 1). Ensuring the high editing efficiency of the CRISPR/Cas system and reducing off-target effects and toxicity to cells is urgently required to study CRISPR/Cas in new species. In this study, the mechanism of the CRISPR/Cas system and its composition are briefly reviewed. This review focuses on three current problems that have limited the development of this system and summarizes optimization strategies, as well as the latest practical application cases, to provide a reference for more researchers in system optimization and new species development.

The versatility of the CRISPR system has given rise to derivative techniques based on nuclear editing, which may contribute to cytotoxicity owing to the unique nature of prokaryotic gene profiling, especially in microorganisms. CRISPR/Cas can lead to fatal chromosomal breaks, resulting in inefficient transformation and gene editing failure (Jing Zhao et al., 2020). In this review, four methods to mitigate Cas9 toxicity are discussed (Figure 2): substituting for Cas proteins with lower toxicity (McNeely et al., 2010); using endogenous immune systems in prokaryotes to relieve the toxicity, and low transformation efficiency of Cas9 (Luo et al., 2015; Wang et al., 2016; Qin et al., 2021); reducing the cytotoxicity caused by DSBs by introducing the non-homologous end joining (NHEJ) repair mechanism (Huang et al., 2019; Su et al., 2019); and emerging novel CRISPR gene editing based on transposons (Strecker et al., 2019).

Although the CRISPR-Cas9 system from Streptococcus pyogenes is widely used in bacterial genome editing, its toxicity has limited research on Streptomycetes (Yeo et al., 2019) and Corynebacterium glutamicum (Jiang et al., 2017). Thus, establishing CRISPR systems in industrial microorganisms by substituting Cas proteins with an alternative with reduced toxicity is essential. Cyanobacterium has been successfully designed and transformed into a strain capable of producing biomass fuels and bioactive substances, such as alkanes and terpenoids (McNeely et al., 2010). However, Cas9 cytotoxicity has hindered the application of the CRISPR/Cas system in cyanobacteria. Unlike Cas9, Cas12a (Cpf1), which is modulated by the lac promoter, can thrive both in crRNA processing and target gene cleavage, resulting in sticky ends (Pattharaprachayakul et al., 2020). Cas12a provides an effective solution for cyanobacteria. However, the reasons for the lower toxicity of Cas12a protein are unknown (McNeely et al., 2010). The same strategy has been applied to Streptomyces, in which the FnCas12a protein from Francisella novicida was found to be effective (Lei Li et al., 2018). Meanwhile, the author found that the CRISPR-FNCas12A3 system of genetically engineered FnCas12a mutant EP16 can recognize the PAM sequence for precise site mutation and insertion. The CRISPR-FNCAS12A3 system solves the limitations of TTN PAM recognition by Streptomyces with a high GC content (Jun Zhang et al., 2020). Additionally, the effector Cas9 (TdCas9_m) from Treponema denticola, Cas9 (NmCas9) from Neisseria meningitides, and Corynebacterium glutamicum codon-optimized Cpf1 (FnCpf1_cg) from Francisella tularensis do not affect cell growth (Sun et al., 2018).

In addition to replacing the exogenous Cas protein, using the endogenous CRISPR/Cas system is also an excellent choice to reduce cytotoxicity and the cellular stress and compatibility caused by the external immune system. Luo et al. first attempted to knockout the Cas3 gene in E. coli K-12 by using the endogenous CRISPR/Cas system to enable target gene expression and multiple regulations. Accidental self-targeting is assumed to be forced in the case of Cas gene deletion-mediated deactivation of the endogenous CRISPR/Cas system. Specifically, this study used Class 1 CRISPR/Cas systems containing Cascade and Cas3 for DNA targeting. The researchers inactivated Cas3 protein and added a constitutive promoter upstream of the cascade operon, which drove transcription to become individual crRNAs, which could participate in binding DNA and insert into the PAM sequence to inhibit transcription (Figure 1B). This study verified the possibility of endogenous CRISPR system development (Luo et al., 2015). Subsequently, based on this principle, Qin et al. constructed a system for the industrial strain Gluconobacter oxydans. They speculated that Cas3 was naturally inactivated by the nucleic acid sequence, and the effectiveness of the system was verified by metabolic engineering. In their study, the endogenous CRISPR interference system (CRISPRi, dCas9 mediated system) was used to study the central carbon metabolic pathway PPP and EDP of G. oxydans, achieving flexible and reliable genome editing of G. oxydans (Qin et al., 2021).

Unlike eukaryotes, most prokaryotes only have an HDR repair mechanism; thus, DSBs caused by the CRISPR/Cas9 system cannot be repaired in cells that have not successfully transformed donor DNA, giving rise to non-viable cells. Therefore, introducing the NHEJ repair mechanism is vital in reducing DSB-induced toxicity. Huang et al. introduced the NHEJ repair pathway by overexpressing the Ku protein and ligase D derived from Mycobacterium in E. coli. They constructed an efficient gene knockout system without donor DNA based on CRISPR/Cas9 and demonstrated for the first time the potential of this system in genetic engineering. The researchers reported that the system was able to delete a chromosome fragment of up to 83 kb with an efficiency of over 85%. Notably, this system does not need to rely on efficient, competent cells, possibly because of its lower cytotoxicity compared to other CRISPR/Cas9 systems (Huang et al., 2019). Similarly, Yan et al. innovatively established genome editing tools that reduced cytotoxicity based on CRISPR cutting and NHEJ repair pathways. Ultimately, this resulted in the formation of deletion mutants in Mycobacterium tuberculosis, wherein the system had access to double mutations simultaneously, demonstrating its potential for screening a large-scale targeted point in drugs (Yan et al., 2020). In addition to Ku protein and ligase D, Su et al. (2019) found that T4 phage ligase could efficiently repair the NHEJ system alone, enabling E. coli to achieve a higher survival rate after producing DSB (Figure 2C).

The transposon is a class of DNA sequences that can be transcribed or reverse transcribed in the genome. The present study has found that a transposase from Scytonema Hofmanni can associate with the CRISPR effector (Cas12k) to form CRISPR-associated transposase (CAST). CAST system utilized Cas12k to identify and bind to specific sites in the genome, and directly inserts exogenous gene fragments into target sites by transposase. Cas12k in this system does not revolve in the cleavage of DNA and homologous recombination due to the absence of endonuclease activity. Experimental results showed that the system could successfully insert 2.5 kb DNA fragments at the target site and the success rate is up to 80%, which was far better than the traditional CRISPR system based on HDR (Strecker et al., 2019). Similarly, Klompe et al. also found the Tn7-like transposon in Vibrio cholerae. They found that after Cascade recognized the target DNA, Cascade directly combined with the protein (TniQ) to guide the transposon into the target site in the genome. After many experiments, transposons were accurately and completely delivered to the target site of the bacterial genome (Klompe et al., 2019).

Currently, the CRISPR/Cas system has been widely developed and applied in commonly studied microorganisms. However, due to some inherent advantages of this system, its development in unconventional hosts is also being carried out based on the three main components (e.g., Cas, gRNA, and donor DNA) required by CRISPR/Cas system. In this context, the system construction strategies can be divided into single-vector expression, multiple-vector expression, and chromosome integration.

Single-vector expression is the most common construction strategy of the CRISPR/Cas gene-editing system, that is, the integration of Cas, gRNA, and donor DNA into the same vector. This strategy has been widely used in the early development of the system combined with GoldnGate molecular cloning techniques, such as CPEC, and has allowed for the shortening of editing cycles. However, its defects lie in the large size of the entire plasmid, which imposes a sizeable physiological burden on cells. Additionally, the plasmid must be imported repeatedly after each round of editing, which requires an effective competent cell. To address this, Huang et al. utilized two promoters (P-thl and P-araE) to express Cas9 and sgRNA in Clostridium ljungdahlii, with deletion efficiencies of up to 100% (Huang et al., 2016). Similarly, Xu et al. (2015) developed Streptococcus pyogenes CRISPR/Cas9 to edit the genome of Clostridium cellulolyticum, which contributed to a high editing efficiency, even when using 200 bp homologous arms.

The multi-vector expression system is used to express CRISPR/Cas components through multiple plasmids, which partly solves the problem of a single-vector expression strategy. Cas can exist in cells for a long time under the control of an inducible promoter. Only gRNA and donor DNA must be constructed for each round of editing, thus addressing the problem of large plasmids in the single-vector strategy, thereby increasing the flexibility of the system. In addition to the two-plasmid system, a strain with many plasmids, such as E. coli, can be used to establish a three-plasmid system. Zhu et al. developed a three-plasmid system for Cas9, gRNA, and donor DNA expressed individually, facilitating plasmid construction because of the independent expression vectors. The system also showed high efficiency in multi-site editing because of the continuous expression of the donor DNA (Zhu et al., 2017). Recently, Kozaeva et al. constructed a group of plasmids based on CRISPR and dCas9 in Pseudomonas putida that could increase the expression of gltA encoding citrate synthase. As a result, accA, an essential gene encoding the subunit of acetyl-CoA carboxylase complex A, was dynamically reduced. The content of acetyl-CoA reconnection was increased by eight times (Kozaeva et al., 2021). Because such multiple plasmids editing systems are limited by the number of plasmids available in the strains, their development in unconventional microorganisms has yet to be fully developed.

The chromosome integrated strategy shows potential for developing strains that do not have multiple available plasmids. However, this strategy involves integrating the Cas gene and gRNA into chromosomes, which requires a traceless operation because of its complex process. In particular, if an iterative cycle system is formed, it is generally necessary to introduce a traceless operating system, leading to a lower prevalence. However, the chromosome integration strategy also has unique advantages, including more stable Cas gene expression, which is less likely to be lost, and the fact that the physiological burden on bacteria is small. In a classic experiment, Westbrook et al. constructed a CRISPR/Cas9 gene-editing box strategy in Bacillus subtilis and integrated spCas9 and gRNA expression frames into lacA and thrC sites of chromosomes, respectively, followed by providing dsDNA as a donor DNA template to achieve genome editing. Notably, the researchers achieved high single-point editing efficiency and induced the simultaneous deletion of double genes in the genome, with an efficiency of up to 85% (Westbrook et al., 2016).

In recent years, genome-editing relying on CRISPR/Cas and its derivative technologies, such as precise editing, multi-gene editing, and precise regulation, has experienced rapid innovation, giving rise to comprehensive solutions and greater flexibility in the development of synthetic biology. In turn, this has allowed for large-scale studies of genes and mutations. However, its development has been hampered by issues with editing efficiency due to off-target effects and cytotoxicity, as well as multiple editing efficiency.

The CRISPR/Cas system may be improved via optimization. To this end, a range of techniques have been applied to detect unexpected mutations to improve in-target efficiency and reduce or avoid off-target effects. Additionally, the development of mutants and analogs of Cas9 also represents an effective method to avoid off-target effects, reduce toxicity, and improve editing efficiency. Using ACR competitive binding, the Cas protein can regulate CRISPR expression. Furthermore, selecting ssDNA rather than dsDNA as the donor DNA can ensure the stable existence of fragments and enable a higher editing efficiency. Furthermore, the rise of DNA base editors without DSBs has allowed researchers to solve the problems associated with the original system.

Researchers have continued to focus on improving the CRISPR/Cas system. A recent report found that CRISPR/Cas9 editing produces micronuclei, and chromosomes are structurally deficient, thus initiating a mutation process known as chromothripsis. Chromothripsis is an extensive chromosomal rearrangement limited to one or more chromosomes, causing congenital diseases and cancer in humans. This suggests that chromothripsis is another manifestation of the off-target effects of CRISPR/Cas9 (Leibowitz et al., 2021). Gene-editing technology has been widely applied in microbial synthetic biology and can quickly, efficiently, and accurately create more chassis organisms suitable for the production of high value-added products. CRISPR gene-editing technology may also contribute to the end of the coronavirus pandemic. Synthetic biology and CRISPR/Cas technology are emerging disciplines, albeit with several unresolved issues. However, with the rapid development and in-depth research of these two complementary technologies, improved applications are likely to be obtained in the near future.