- Mini Review
- Open access
- Published:
Current advances in CRISPR-Cas-mediated gene editing and regulation in cyanobacteria
Blue Biotechnology volume 1, Article number: 9 (2024)
Abstract
Photosynthetic cyanobacteria are important microbial models in basic research such as photosynthesis, biological rhythm, and the geochemical cycle of elements. Meanwhile, they attract significant attention to serve as "autotrophic cell factories", enabling the production of dozens of chemicals. In this case, genetic toolboxes especially gene editing and regulation tools with high efficiency are the basis of the development of related studies. Among them, clustered regularly interspaced palindromic repeats (CRISPR)-Cas related technologies have realized rapid and efficient gene editing, gene silence and activation in multiple organisms like Escherichia coli, budding yeast, plant and mammalian cells. To promote their understandings and applications in cyanobacteria, in this review, advances in CRISPR-Cas-mediated gene editing and regulations were critically discussed. Firstly, the elucidation of native CRISPR-Cas in cyanobacteria were concluded, which provided new tool candidates for further optimization. Secondly, basic principles and applications of CRISPR-Cas related gene editing and regulation tools used in cyanobacteria were respectively discussed. In the future, further studies on development of native CRISPR-Cas tools, continuous editing and dynamic regulation would significantly promote the synthetic biology researches in cyanobacteria.
Introduction
In the context of a low-carbon economy, cyanobacteria, as photosynthetic microorganisms, have garnered considerable attention for their ability to produce high-value-added chemicals from sustainable resources such as water, sunlight, CO2, and mineral salts. Compared to land plants, cyanobacteria exhibit higher CO2 fixation efficiency, attributed to the presence of a carbon concentration mechanism [1], which offers significant potential for achieving negative carbon production of chemicals and bioactive compounds. Leveraging synthetic biology approaches, cyanobacteria have successfully synthesized numerous high-value-added chemicals, including biofuels, bioplastics, bioactive compounds [2, 3], biologics [4, 5] and agents for environmental governance [6,7,8]. However, the efficient advancement of cyanobacteria-related research relies heavily on the availability of efficient gene manipulation tools.
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, widely recognized as adaptive immune systems in archaea and bacteria [9], are also prevalent in cyanobacteria. They have garnered significant attention due to their high efficiency, precision, and programmable characteristics for gene editing, and have found applications in various fields such as such as gene therapy [10], diagnosis [11], screening [12], and metabolic engineering [13]. Building on CRISPR-Cas9 and Cas12a, a plethora of genetic tools has been developed in cyanobacteria, including base editing [14], gene inhibition [15], gene activation [16], and genome large fragment knockout [17]. The exploration of endogenous CRISPR-Cas systems in cyanobacteria, coupled with the development of genetic tools based on these systems, has led to significant advancements. For instance, the Cas12k effector from the filamentous cyanobacterium Scytonema hofmannii was employed to generate homozygotes following the integration of target genes into the genome of Anabaena sp. PCC 7120 [18]. A wide variety of genetic tools based on the CRISPR-Cas systems have provided avenues for basic research and engineering in cyanobacteria.
To promote their understandings and applications in cyanobacteria, in this review, advances in CRISPR-Cas-mediated gene editing and regulations were discussed. Detailly, the elucidation of native CRISPR-Cas in cyanobacteria were first concluded, which provided new tool candidates for further optimization. Then, basic principles and applications of CRISPR-Cas related gene editing and regulation tools used in cyanobacteria were respectively discussed, which were the main focus of this article. Finally, future directions including development of native CRISPR-Cas tools, continuous editing and dynamic regulation were presented. The article here provides new insights for CRISPR-Cas related studies in cyanobacteria.
Native CRISPR-Cas systems in cyanobacteria
The adaptive immune process of the CRISPR-Cas system is mainly divided into three processes: foreign gene acquisition, crRNA biogenesis, and target interference [19] (Fig. 1). At the foreign DNA acquisition stage, a complex of specific Cas proteins binds to the invading target DNA to obtain fragments of 30–50 bases in length, known as the protospacers [19]. Adjacent to this sequence, there is a specific motif sequence recognized by the Cas proteins, called the protospacer adjacent motif (PAM) [20]. After the 5' end of the CRISPR repeat sequence is replicated, protospacers are inserted into the CRISPR array with the assistance of Cas1 and Cas2 proteins forming the spacer sequence [21]. At the crRNA biogenesis stage, single-stranded RNA transcribed by a CRISPR array, known as pre-CRISPR RNA (pre-crRNA), is processed into crRNA after undergoing cleavage by CRISPR-specific endoribonucleases [20]. These endoribonucleases vary across different CRISPR-Cas systems and may consist of multi-protein Cas complexes, single multi-domain Cas proteins, or non-Cas host RNAs [22]. At the target interference stage, crRNAs often remain bound to the processing complex and serve as guides to recognize invading DNA, which is subsequently degraded by Cas nucleases (or nuclease) [22].
The CRISPR-Cas system consists of a CRISPR array and Cas protein(s). The CRISPR array produces crRNA, which guides the effector composed of Cas protein(s) to cleave and degrade invading DNA or RNA. By analyzing the genetic composition and locus structure of the CRISPR-Cas systems, they have been divided into 2 classes, 6 types and 33 subtypes [22]. Class 1 systems (Type I, Type III, and Type IV) have effector modules composed of multiple Cas proteins, some of which form complexes mediate pre-crRNA maturation that, with contribution from additional Cas proteins, mediate interference [22,23,24] (Fig. 1). By contrast, class 2 systems (Type II, Type V, and Type VI) have a single multi-domain Cas protein that combines all the features for mature pre-crRNA and interference [22,23,24] (Fig. 1).
CRISPR-Cas systems are widely distributed among cyanobacteria, and Type I and Type III CRISPR-Cas systems are extremely abundant [25,26,27]. However, the CRISPR-Cas system was almost completely lost in marine cyanobacteria, despite being in an algae-rich environment, which is a primary driving force for the widespread distribution of CRISPR-Cas systems [25, 28, 29]. Additionally, the type I-F, I-B, and V-K systems, utilized by bacterial transposons of the Tn7 family to guide RNA-directed transposon insertion, have been identified in cyanobacteria [30, 31].
Class 1 CRISPR-Cas systems
Three CRISPR-Cas systems located in the pSYSA plasmid were identified in Synechocystis sp. PCC6803 (hereafter Syn6803), they belong to Type I-D, Type III, and Type-III-B, respectively [32,33,34]. The Type I-D and Type III systems contain Cas6 protein related to pre-crRNA processing, respectively, while the Type III-B system lacks this important protein, however, each of them functions independently [33]. Synechocystis sp. PCC6714, which was separated from the same environment as Syn6803, possesses a Type III-B system that is highly homologous to the one found in Syn6803 [32]. This may result from the horizontal transfer of genes. The Type III system mostly uses the Cas6 protein provided in trans by other CRISPR-Cas loci for pre-RNA processing but is not found in the Type III-B system of Syn6803, which uses endogenous RNase E for pre-crRNA processing [22, 33, 34].
There are abundant Type I-A, I-D, III-A, and III-B systems in Microcystis aeruginosa, and the direct repeat sequences (DRs) of the same type have similar secondary structures, and the existence of different and special secondary structures play an important role in the recognition of Cas proteins, and in addition, except for the DRs associated with Type III-A, all DRs have the same 3’ sequence [26]. The foreign DNA acquisition stage of the CRISPR-Cas system may involve a conserved mechanism. The spacer sequences in the CRISPR array display homologous fragments with the sequences of bacteria (including cyanobacteria), plasmids, and viruses, implying that the CRISPR-Cas systems confer resistance against mobile genetic elements, plasmids, and viruses [27].
In Anabaena (Nostoc) sp. PCC 7120 (hereafter Ana7120), there are 3 different Class 1 systems (Type I-A, Type I-D, and Type III-D) and 1 Class 2 (Type V-K) system, containing 11 DRs, all capable of producing mature crRNAs [27]. Some of the DRs may belong to the residual CRISPR-Cas system and be able to play a partial role, although not experimentally verified. In the Type I-D system, the backbone protein Cas7 has been shown to have structural domains that bind to specific crRNAs [35]. This strongly suggests that the system is biologically functional with endogenous sources. The Cas6 protein of Type I-D cross-talks in recognizing DRs and can cleave DR sequences specifically recognized by Type I-A, a process that would limit the accumulation of crRNA in the Type I-D system [36]. Besides, the Type V-K effector protein All3613 may only be one subunit of the Cas12k protein [22, 27]. This type of CRISPR-Cas system is rare in cyanobacteria.
In a study of the CRISPR-Cas systems associated with transposition in cyanobacteria, an abundance of Tn7-like transposon genes was found in cyanobacteria, and a novel Type I-D system was identified [31]. In the classic Type I-D system, Cas1 and Cas2 proteins are involved in spacer acquisition, while Cas3 protein is responsible for unwinding double-stranded DNA, despite having separate functional domains [37]. However, in the novel Type I-D system, the Cas1, Cas2, and Cas3 proteins are missing, which is a conservative phenomenon in transposon-associated CRISPR-Cas systems [31]. The novel Type I-D system, derived from Myxacorys californica WJT36-NPBG1, is biologically functional in Escherichia coli [31], whereas the Type I-D system derived from M. aeruginosa has been utilized for gene editing in mammalian cells [38].
Class 2 CRISPR-Cas systems
Class 2 CRISPR-Cas systems are relatively uncommon in cyanobacteria [25,26,27], with only Type V-K CRISPR-Cas systems discovered thus far [18, 27, 30]. Currently, there is a lack of direct evidence suggesting full activity in the Type V-K system in Ana7120, and its potential effector protein, All3613, is also likely to be only one of the subunits that make up the Cas12k core. The functionality of the Type V-K system derived from Scytonema hofmannii has been experimentally validated and applied in human cells as well as in Ana7120 [18, 30]. The Type V-K system consists of four components: the pseudonuclease Cas12k, the transposase TnsB, the AAA + ATPase TnsC, and the zinc finger protein TniQ [39]. The functioning process of this system involves the complexation of crRNA with Cas12K, guided by crRNA, recognizing and binding to the target DNA [30]. TnsC accumulates by interacting with Cas12k bound to the double-stranded DNA [30]. With the assistance of ATP, TnsC polymerizes to form a helical filament [30]. Subsequently, upon binding of TniQ to the helical filament, the generation of the filament is inhibited [30]. The filament serves as a platform for recruiting TnsB [30]. After recruiting TnsB, which carries the transposon, and completing the transposition process, the ATPase activity of TnsC is activated by interaction with TnsB [30]. This activation leads to the degradation of the helical filament [30]. Thus, the entire RNA-mediated transposition process is completed.
Off-target events may occur during the transposition process mediated by the Type V-K system, particularly after an increase in TnsC expression levels [39]. The occurrence of non-specific transposition events is primarily driven by TnsC, attributed to its preference for AT-rich regions in DNA binding. While currently suppressing non-specific transposition events by controlling the expression level of TnsC, this method does not fundamentally address the issue and still poses a high risk of off-target effects, thus limiting the universality of the system.
CRISPR-Cas mediated gene editing in cyanobacteria
Overview of CRISPR-Cas mediated gene editing tools
DNA fragment editing
Gene editing, which involves the insertion, deletion, or replacement of DNA fragments in the genome, is referred to here as DNA fragment editing. DNA fragment editing technology based on the CRISPR-Cas system relies on the recognition and cleavage of target sequences by the crRNA-effector complex, followed by repair by the organism's own DNA repair mechanisms [39]. To ensure the accuracy of repair outcomes, donor fragments containing homologous sequences are often provided (Fig. 2A).
CRISPR-Cas9 and CRISPR-Cas12a are the main CRISPR-Cas systems used for DNA fragment editing [39, 40]. While Cas9 exhibits higher toxicity in some species [41, 42], Cas12a demonstrates greater universality in cyanobacteria [43]. Cas12a possesses both endoribonuclease and endonuclease activities, enabling it to independently process pre-crRNA into mature crRNA [44]. In the CRISPR-Cas12a DNA fragment editing tool, after processing pre-crRNA into mature crRNA, Cas12a forms a complex with the crRNA to recognize and cleave the target sequence, resulting in double-strand breaks. The broken DNA can be repaired by either non-homologous end joining (NHEJ), resulting in random insertions or deletions, or homology-directed repair (HDR) mechanisms, which can generate precise editing outcomes when provided with a homologous repair template, within the cell [45, 46] (Fig. 2A). Another CRISPR-Cas3 tool developed based on Class I can degrade large fragments bidirectionally under the action of Cas3 helicase and exonuclease activities, without the need for a repair template [47] (Fig. 2A). This tool is able to cleave the recognition site bidirectionally, resulting in editing outcomes with different deletion lengths.
Base editing
Base editing entails modifying the bases of the genome. In classical base editing technology, a deaminase enzyme specific to the target base is fused with an effector protein. Under the guidance of crRNA, the fused effector complex targets the desired sequence and catalyzes the biochemical reaction to modify the corresponding base [48,49,50,51,52] (Fig. 2B).
The classical base editing tool is based on Class 2 CRISPR-Cas systems, where cytidine deaminase or adenine deaminase is fused to the C-terminal or N-terminal of the Cas protein. Cytidine deaminase converts C to U after deamination, then the copied DNA strand replaces U with T using the DNA containing U as a template, completing the C → T base editing process (CBE) [52]. Adenine deaminase converts A to inosine after deamination (A → I), then inosine is repaired to G, completing the A → G base editing process (ABE) [49]. Moreover, by fusing adenine deaminase and cytidine deaminase or mutating adenine deaminase, a dual-function base editor is developed based on CBE and ABE [53, 54]. Building upon CBE, uracil DNA glycosylase (UDG) is used to recognize and cleave the U base, generating a DNA strand without a base [55]. During the repair process, different species exhibit different preferences. For example, in Escherichia coli, primarily C → A results are produced, while in mammalian cells, primarily C → G results are observed, showing considerable randomness [55, 56]. Therefore, in CBE, a uracil glycosylase inhibitor is typically introduced to enhance the efficiency of CBE base editing outcomes [52]. In cyanobacteria, Type IV UDG is not inhibited by phage-derived UGI [57]. Although using CRISPRi to suppress UDG expression has improved the editing efficiency of CBE, it has also increased the complexity of the CBE system, making it not an ideal solution [57]. An ideal approach would be to find a UGI that can inhibit UDG activity in cyanobacteria. However, it is not necessary to be overly concerned about the impact of UDG activity on the CBE tool. Another study using the CBE tool in Synechococcus elongatus PCC 7942 (hereafter Syn7942) demonstrated that the CBE tool still exhibited very high editing efficiency without inhibiting UDG activity [14].
Due to the absence of an amino group in the T base, base editing tools based on deaminase cannot directly edit the T base. To expand the types of base editing, a base editing tool based on DNA glycosylase has been developed [58, 59]. DNA glycosylase creates an apurinic/apyrimidinic (AP) site at the target location, triggering the base excision repair mechanism, thereby achieving the goal of base editing [60]. While base editing tools based on DNA glycosylase have broadened the types of base editing, their use is limited due to the randomness of repair outcomes associated with the base excision repair mechanism. Currently, there have been no reports of the development of base editing tools based on glycosylase in cyanobacteria. This represents one of the future directions for the development of base editing tools in cyanobacteria.
Applications of CRISPR-Cas systems for gene editing in cyanobacteria
DNA fragment editing in cyanobacteria
DNA fragment editing tools based on CRISPR-Cas12a have been successfully developed and applied in cyanobacteria. In Ana7120, under the "two spacers" strategy, the editing efficiency reached 100%, with a maximum editing length of 118 kb [61]. Furthermore, by introducing the sucrose-sensitive counter-selection gene sacB onto the vector, rapid removal of edited plasmids was achieved [61]. Additionally, using this tool, the RBS sequence of the key gene polA, encoding DNA polymerase I, was replaced in Ana7120 [61], providing a powerful tool for studying critical genes in cyanobacteria. Using DNA fragment editing tools developed based on CRISPR-Cas12a, successful deletions of predicted non-essential genes in Syn7942 have been achieved, along with the combination deletion of non-essential genes at different loci [17].
In Synechococcus elongatus UTEX 2973 (hereafter Syn2973), there have also been reports of using CRISPR-Cas3 for the deletion of non-essential genes [62]. Interestingly, Syn 2373, after deleting non-essential genes, exhibited advantages in growth and sucrose production, a phenomenon not observed in the study of non-essential gene deletion in Syn7942 [17, 62]. This could be due to the greater flexibility in deletion lengths generated by the CRISPR-Cas3 system. In the study of Syn2973's response to high light tolerance by truncating the light-harvesting antenna, strains after knocking out the gene encoding the rod-core linker gene cpcG based on CRISPR-Cas12a did not show an advantage [63]. However, targeting the gene encoding the rod-rod linker gene cpcC2 using the CRISPR-Cas3 editing tool resulted in a mutant strain with the deletion of 3 rod-rod linkers (cpcC1, cpcC2, cpcD) and a phycocyanin gene cpcB2A2, exhibiting high-light tolerance characteristics [63]. In rational DNA fragment editing, systems based on CRISPR-Cas12a have an advantage, while in non-rational DNA fragment editing, systems based on CRISPR-Cas3 often yield some exciting results.
Base editing in cyanobacteria
CBE is used for gene silencing in Syn7942 by modifying the codons in the target gene coding region to stop codons, leading to premature termination of translation of the target gene [14]. Premature termination base editing of the glgP gene encoding glycogen phosphorylase and the glgX gene encoding glycogen debranching enzyme resulted in increased accumulation of glycogen [14]. Base editing tools have been less reported in cyanobacteria, and applications of other forms of base editing tools in cyanobacteria have not been widely observed yet.
CRISPR-Cas mediated gene regulation in cyanobacteria
Mechanisms of CRISPR-Cas mediated gene regulation
CRISPR interference (CRISPRi) primarily relies on catalytically inactive forms of dCas9 and dCas12a, guided by crRNA to bind to target genes, thereby interfering with the transcription process and reducing the expression levels of the target genes [64, 65] (Fig. 3A). By introducing multiple sgRNA/crRNA sequences, it is possible to achieve simultaneous suppression of multiple genes or achieve multiplexed suppression of the same gene, meeting the requirements for controlling gene expression [66] (Fig. 3B). In addition, CRISPRi can screen key genes on a genome-wide scale by synthesizing gRNA libraries [67].
Applications of CRISPR-Cas systems for gene regulation in cyanobacteria
CRISPRi-based gene regulation
To increase the production of succinic acid in Syn7942, the CRISPRi tool based on dCas9 was employed to individually inhibit three key genes: glgC, associated with sucrose accumulation, and sdhA and sdhB, associated with succinic acid decomposition metabolism [68]. All three interventions resulted in increased accumulation of succinic acid [68]. To further enhance the yield of succinic acid, in addition to overexpressing genes related to the succinic acid synthesis pathway, genes glgC, and sdhB were simultaneously inhibited using the CRISPRi tool based on dCas9, resulted in a final yield increase of 82% [69]. In Synechocystis sp. PCC 6803 (Syn6803), it has also been demonstrated that the CRISPRi tool based on dCas9 is effective for simultaneously inhibiting multiple genes [15]. In Syn6803, the CRISPRi tool based on dCas9 was utilized to identify genes associated with extracellular polymeric substance (EPS) synthesis [70]. By individually inhibiting three candidate genes—slr0977, slr2107, and sll0574—it was determined that the slr0977 gene is a key gene involved in EPS synthesis [70].
Compared to the CRISPR-Cas9 system, Cas12a has the ability to independently process pre-crRNA containing spacer-direct repeats into crRNA, giving it an advantage in multi-gene inhibition [44, 71]. In Syn7942, the CRISPRi system based on CRISPR-Cas12a, utilizing a single crRNA array, can achieve the inhibition of three genes [72]. Moreover, the strength of inhibition does not change with an increase in the number of genes targeted for inhibition [72]. Furthermore, increasing the yield of β-ionone has been achieved through the inhibition of the gene encoding aconitate hydratase, acnB, or the gene encoding the phycocyanin β-subunit, cpcB2 [72]. It is worth noting that growth inhibition of the strain occurred during the inhibition of genes acnB or cpcB2, despite the use of a lactose operon to control the expression of dCas12a. However, the rigor of the induction system was not rigorously assessed in the relevant study. In Syn2973, utilizing a lactose operon to control dCas12a and the crRNA array, the expression of target genes was inhibited by 0–10% under non-induced conditions [73]. However, upon the addition of the inducer, the inhibition of target genes could reach over 90% [73]. The rigor of the induction system controlling the CRISPRi system remains an important issue that needs to be addressed. In Syn6803, controlling the expression of dCas12a with a riboswitch responsive to theophylline and controlled by a rhamnose inducible promoter, when inhibiting the gene psbD encoding the PSII reaction center protein D2, there was no effect on strain growth under non-induced conditions [74]. However, when only using the rhamnose-inducible promoter to control dCas12a, significant growth inhibition of the strain was observed under non-induced conditions [74]. A rigorous CRISPRi system provides a good solution for studying essential genes in cyanobacteria.
Screening library in cyanobacteria
Biochips enable the rapid synthesis of guide RNA libraries at a whole-genome scale, offering the possibility for the establishment of whole-genome scale CRISPRi libraries. The CRISPRi library can be coupled with growth under different conditions for screening, and it can also be combined with microfluidic techniques for screening. In cyanobacteria, construction of a whole-genome scale CRISPRi library has only been completed in Syn6803 so far [75]. Under L-lactate stress as the screening condition, after continuous cultivation of the CRISPRi library, a significant increase was identified in the bcp2 mutant encoding the bacterioferritin comigratory protein [75]. Combining microfluidic techniques for screening the CRISPRi library, mutants with high L-lactate production were identified. The repression of gene gltA (citrate synthase) or pcnB (CCA-tRNA nucleotidyltransferase) resulted in significantly increased L-lactate yield [75].
Future direction
The development and utilization of endogenous CRISPR-Cas systems
Analysis of CRISPR-Cas systems in cyanobacteria with reference genomes has revealed their widespread distribution, indicating a rich resource of CRISPR-Cas systems within cyanobacteria [25,26,27]. Research on endogenous CRISPR-Cas systems in cyanobacteria such as Syn6803, Ana7120, and M. aeruginosa has reached a relatively mature stage [26, 31,32,33,34, 36, 76]. However, there are still numerous CRISPR-Cas resources waiting to be discovered. Further development of endogenous CRISPR-Cas systems in cyanobacteria into genetic manipulation tools still has a long way to go. It's worth noting that CRISPR-Cas systems, such as Type I-D from M. aeruginosa and Type V-K from Scytonema hofmannii, have been applied in genetic editing of other species [18, 38]. The evidence suggests that CRISPR-Cas systems sourced from cyanobacteria are versatile and have the potential to be developed into genetic manipulation tools applicable to various host cells.
Continuous editing of cyanobacterial genomes
Achieving continuous multi-step gene editing in host cells is a fundamental requirement for realizing artificial cell factories. Editing the cyanobacterial genome under untagged conditions involves removing the plasmid containing the editor after editing is completed. Currently, the approach involves introducing the sacB gene into the plasmid carrying the editor [61]. After the editing process is completed, the plasmid is removed by adding sucrose [61]. The characteristic of multiple copies in the cyanobacterial genome raises concerns about whether the genome can maintain the edited state stably after plasmid removal. Although tools have been developed to obtain homozygous mutants in cyanobacteria [18], the CRISPR-Cas systems used in these tools are relatively complex, which limits their flexibility. Therefore, it is necessary to develop a flexible gene editing tool that couples plasmid removal with homozygous screening.
Dynamic regulation
During the process of regulating genes in engineered strains to enhance product yield, premature inhibition often affects the biomass of the strains. Such phenomena have been observed during the use of CRISPRi to regulate genes in cyanobacteria [72]. To ensure dynamic regulation of target genes, strict induction system control over the expression of the CRISPRi system is necessary. In cyanobacteria, various control systems such as lactose operon, dehydrotetracycline inducible system, and theophylline-responsive RBS have been used to regulate the CRISPRi system. However, in related reports, instances of inhibitory effects on target genes under non-inducing conditions still exist [72, 74]. Therefore, the impact of the CRISPRi system on target genes under non-inducing conditions, as well as the degree of inhibition of target genes under induced conditions, is crucial for dynamic regulation. Currently, there hasn't been a systematic study on the dynamic range of dynamic regulation systems in cyanobacteria.
Conclusion
CRISPR-Cas systems have emerged as powerful tools for genetic manipulation in cyanobacteria. With their remarkable diversity, cyanobacteria present an extensive reservoir ripe for further exploration. As research into endogenous CRISPR-Cas systems in cyanobacteria advances, applications of these systems in genetic manipulation tools are beginning to surface. Utilizing Cas9 and Cas12a, tools have been developed enabling seamless deletion of large genomic segments, single-base modifications, and transcriptional level gene suppression in cyanobacteria, thereby propelling the realization of cyanobacterial cell factories. The development of CRISPRi libraries has expedited the screening of genes associated with specific phenotypes. Nevertheless, there are still areas for advancement in CRISPR-Cas-based genetic manipulation tools in cyanobacteria, including continuous gene editing, generation of homozygous mutants, removal of editing cassettes, ensuring the precision and induction levels of induction systems for dynamic regulation.
Availability of data and materials
No datasets were generated or analysed during the current study.
Abbreviations
- Ana7120:
-
Anabaena (Nostoc) sp. PCC 7120
- ABE:
-
A → G base editor
- CBE:
-
C → T base editor
- CRISPR/Cas:
-
Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins
- DRs:
-
Direct repeats
- HDR:
-
homology-directed repair
- PAM:
-
Protospacer adjacent motif
- NHEJ:
-
Non-homologous end joining
- pre-crRNA:
-
pre-CRISPR RNA
- Syn6803:
-
Synechocystis Sp. PCC6803
- Syn7942:
-
Synechococcus elongatus PCC 7942
- Syn2973:
-
Synechococcus elongatus UTEX 2973
- UDG:
-
Uracil DNA glycosylase
- AP:
-
Apurinic/Apyrimidinic site
References
Hagemann M, Kern R, Maurino VG, Hanson DT, Weber APM, Sage RF, et al. Evolution of photorespiration from cyanobacteria to land plants, considering protein phylogenies and acquisition of carbon concentrating mechanisms. J Exp Bot. 2016;67(10):2963–76. https://doi.org/10.1093/jxb/erw063.
Abed RMM, Dobretsov S, Sudesh K. Applications of cyanobacteria in biotechnology. J Appl Microbiol. 2009;106(1):1–12. https://doi.org/10.1111/j.1365-2672.2008.03918.x.
Zahra Z, Choo DH, Lee H, Parveen A. Cyanobacteria: Review of Current Potentials and Applications. Environments. 2020;7(2):13. https://doi.org/10.3390/environments7020013.
Sun T, Zhang Y, Zhang C, Wang H, Pan H, Liu J, et al. Cyanobacteria-Based Bio-Oxygen Pump Promoting Hypoxia-Resistant Photodynamic Therapy. Frontiers in Bioengineering and Biotechnology. 2020;8. https://doi.org/10.3389/fbioe.2020.00237.
Zhang X, Zhang Y, Zhang C, Yang C, Tian R, Sun T, et al. An injectable hydrogel co-loading with cyanobacteria and upconversion nanoparticles for enhanced photodynamic tumor therapy. Colloids Surf, B. 2021;201: 111640. https://doi.org/10.1016/j.colsurfb.2021.111640.
Duan H, Liu W, Zhou L, Han B, Huo S, El-Sheekh M, et al. Improving saline-alkali soil and promoting wheat growth by co-applying potassium-solubilizing bacteria and cyanobacteria produced from brewery wastewater. Front Environ Sci. 2023;11. https://doi.org/10.3389/fenvs.2023.1170734.
El-Sheekh M, El-Dalatony MM, Thakur N, Zheng Y, Salama E-S. Role of microalgae and cyanobacteria in wastewater treatment: genetic engineering and omics approaches. Int J Environ Sci Technol. 2022;19(3):2173–94. https://doi.org/10.1007/s13762-021-03270-w.
Ahmad IZ. The usage of Cyanobacteria in wastewater treatment: prospects and limitations. Lett Appl Microbiol. 2022;75(4):718–30. https://doi.org/10.1111/lam.13587.
Pinilla-Redondo R, Russel J, Mayo-Muñoz D, Shah Shiraz A, Garrett Roger A, Nesme J, et al. CRISPR-Cas systems are widespread accessory elements across bacterial and archaeal plasmids. Nucleic Acids Res. 2021;50(8):4315–28. https://doi.org/10.1093/nar/gkab859.
Zhang B. CRISPR/Cas gene therapy. J Cell Physiol. 2021;236(4):2459–81. https://doi.org/10.1002/jcp.30064.
Kaminski MM, Abudayyeh OO, Gootenberg JS, Zhang F, Collins JJ. CRISPR-based diagnostics. Nature Biomedical Engineering. 2021;5(7):643–56. https://doi.org/10.1038/s41551-021-00760-7.
Bock C, Datlinger P, Chardon F, Coelho MA, Dong MB, Lawson KA, et al. High-content CRISPR screening. Nature Reviews Methods Primers. 2022;2(1):8. https://doi.org/10.1038/s43586-021-00093-4.
Zhu H, Li C, Gao C. Applications of CRISPR–Cas in agriculture and plant biotechnology. Nat Rev Mol Cell Biol. 2020;21(11):661–77. https://doi.org/10.1038/s41580-020-00288-9.
Wang S-Y, Li X, Wang S-G, Xia P-F. Base editing for reprogramming cyanobacterium Synechococcus elongatus. Metab Eng. 2023;75:91–9. https://doi.org/10.1016/j.ymben.2022.11.005.
Yao L, Cengic I, Anfelt J, Hudson EP. Multiple Gene Repression in Cyanobacteria Using CRISPRi. ACS Synth Biol. 2016;5(3):207–12. https://doi.org/10.1021/acssynbio.5b00264.
Wang T, Zhu H, Yang C. Development of CRISPRa for metabolic engineering applications in cyanobacteria. Synthetic Biol J. 2023;4(4):824–39. https://doi.org/10.12211/2096-8280.2022-077.
Hou F, Ke Z, Xu Y, Wang Y, Zhu G, Gao H, et al. Systematic Large Fragment Deletions in the Genome of Synechococcus elongatus and the Consequent Changes in Transcriptomic Profiles. Genes. 2023;14(5):1091. https://doi.org/10.3390/genes14051091.
Arévalo S, Pérez Rico D, Abarca D, Dijkhuizen LW, Sarasa-Buisan C, Lindblad P, et al. Genome Engineering by RNA-Guided Transposition for Anabaena sp. PCC 7120. ACS Synthetic Biology. 2024. https://doi.org/10.1021/acssynbio.3c00583.
Carter J, Wiedenheft B. SnapShot: CRISPR-RNA-guided adaptive immune systems. Cell. 2015;163(1):260-. e1. https://doi.org/10.1016/j.cell.2015.09.011
Sorek R, Lawrence CM, Wiedenheft B. CRISPR-Mediated Adaptive Immune Systems in Bacteria and Archaea. Annual Review of Biochemistry. 2013;82(Volume 82, 2013):237–66. https://doi.org/10.1146/annurev-biochem-072911-172315.
McGinn J, Marraffini LA. Molecular mechanisms of CRISPR–Cas spacer acquisition. Nat Rev Microbiol. 2019;17(1):7–12. https://doi.org/10.1038/s41579-018-0071-7.
Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS, Brouns SJJ, et al. Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol. 2020;18(2):67–83. https://doi.org/10.1038/s41579-019-0299-x.
Makarova KS, Haft DH, Barrangou R, Brouns SJJ, Charpentier E, Horvath P, et al. Evolution and classification of the CRISPR–Cas systems. Nat Rev Microbiol. 2011;9(6):467–77. https://doi.org/10.1038/nrmicro2577.
Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, et al. An updated evolutionary classification of CRISPR–Cas systems. Nat Rev Microbiol. 2015;13(11):722–36. https://doi.org/10.1038/nrmicro3569.
Cai F, Axen SD, Kerfeld CA. Evidence for the widespread distribution of CRISPR-Cas system in the Phylum Cyanobacteria. RNA Biol. 2013;10(5):687–93. https://doi.org/10.4161/rna.24571.
Yang C, Lin F, Li Q, Li T, Zhao J. Comparative genomics reveals diversified CRISPR-Cas systems of globally distributed Microcystis aeruginosa, a freshwater bloom-forming cyanobacterium. Frontiers in Microbiology. 2015;6. https://doi.org/10.3389/fmicb.2015.00394.
Hou S, Brenes-Álvarez M, Reimann V, Alkhnbashi OS, Backofen R, Muro-Pastor AM, et al. CRISPR-Cas systems in multicellular cyanobacteria. RNA Biol. 2019;16(4):518–29. https://doi.org/10.1080/15476286.2018.1493330.
Godde JS, Bickerton A. The Repetitive DNA Elements Called CRISPRs and Their Associated Genes: Evidence of Horizontal Transfer Among Prokaryotes. J Mol Evol. 2006;62(6):718–29. https://doi.org/10.1007/s00239-005-0223-z.
Sorek R, Kunin V, Hugenholtz P. CRISPR — a widespread system that provides acquired resistance against phages in bacteria and archaea. Nat Rev Microbiol. 2008;6(3):181–6. https://doi.org/10.1038/nrmicro1793.
Querques I, Schmitz M, Oberli S, Chanez C, Jinek M. Target site selection and remodelling by type V CRISPR-transposon systems. Nature. 2021;599(7885):497–502. https://doi.org/10.1038/s41586-021-04030-z.
Hsieh S-C, Peters JE. Discovery and characterization of novel type I-D CRISPR-guided transposons identified among diverse Tn7-like elements in cyanobacteria. Nucleic Acids Res. 2022;51(2):765–82. https://doi.org/10.1093/nar/gkac1216.
Hein S, Scholz I, Voß B, Hess WR. Adaptation and modification of three CRISPR loci in two closely related cyanobacteria. RNA Biol. 2013;10(5):852–64. https://doi.org/10.4161/rna.24160.
Scholz I, Lange SJ, Hein S, Hess WR, Backofen R. CRISPR-Cas Systems in the Cyanobacterium Synechocystis sp. PCC6803 Exhibit Distinct Processing Pathways Involving at Least Two Cas6 and a Cmr2 Protein. PLOS ONE. 2013;8(2):e56470. https://doi.org/10.1371/journal.pone.0056470.
Behler J, Sharma K, Reimann V, Wilde A, Urlaub H, Hess WR. The host-encoded RNase E endonuclease as the crRNA maturation enzyme in a CRISPR–Cas subtype III-Bv system. Nat Microbiol. 2018;3(3):367–77. https://doi.org/10.1038/s41564-017-0103-5.
Kalwani P, Rath D, Ballal A. Novel molecular aspects of the CRISPR backbone protein ‘Cas7’ from cyanobacteria. Biochemical Journal. 2020;477(5):971–83. https://doi.org/10.1042/bcj20200026.
Reimann V, Ziemann M, Li H, Zhu T, Behler J, Lu X, et al. Specificities and functional coordination between the two Cas6 maturation endonucleases in Anabaena sp. PCC 7120 assign orphan CRISPR arrays to three groups. RNA Biology. 2020;17(10):1442–53. https://doi.org/10.1080/15476286.2020.1774197.
Shmakov S, Smargon A, Scott D, Cox D, Pyzocha N, Yan W, et al. Diversity and evolution of class 2 CRISPR–Cas systems. Nat Rev Microbiol. 2017;15(3):169–82. https://doi.org/10.1038/nrmicro.2016.184.
Osakabe K, Wada N, Murakami E, Miyashita N, Osakabe Y. Genome editing in mammalian cells using the CRISPR type I-D nuclease. Nucleic Acids Res. 2021;49(11):6347–63. https://doi.org/10.1093/nar/gkab348.
Strecker J, Ladha A, Gardner Z, Schmid-Burgk JL, Makarova KS, Koonin EV, et al. RNA-guided DNA insertion with CRISPR-associated transposases. Science. 2019;365(6448):48–53. https://doi.org/10.1126/science.aax9181.
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012;337(6096):816–21. https://doi.org/10.1126/science.1225829.
Jiang W, Brueggeman AJ, Horken KM, Plucinak TM, Weeks DP. Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii. Eukaryot Cell. 2014;13(11):1465–9. https://doi.org/10.1128/ec.00213-14.
Jiang Y, Qian F, Yang J, Liu Y, Dong F, Xu C, et al. CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nat Commun. 2017;8(1):15179. https://doi.org/10.1038/ncomms15179.
Ungerer J, Pakrasi HB. Cpf1 Is A Versatile Tool for CRISPR Genome Editing Across Diverse Species of Cyanobacteria. Sci Rep. 2016;6(1):39681. https://doi.org/10.1038/srep39681.
Fonfara I, Richter H, Bratovič M, Le Rhun A, Charpentier E. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature. 2016;532(7600):517–21. https://doi.org/10.1038/nature17945.
Rulten SL, Grundy GJ. Non-homologous end joining: Common interaction sites and exchange of multiple factors in the DNA repair process. BioEssays. 2017;39(3):1600209. https://doi.org/10.1002/bies.201600209.
Shrivastav M, De Haro LP, Nickoloff JA. Regulation of DNA double-strand break repair pathway choice. Cell Res. 2008;18(1):134–47. https://doi.org/10.1038/cr.2007.111.
Csörgő B, León LM, Chau-Ly IJ, Vasquez-Rifo A, Berry JD, Mahendra C, et al. A compact Cascade–Cas3 system for targeted genome engineering. Nat Methods. 2020;17(12):1183–90. https://doi.org/10.1038/s41592-020-00980-w.
Komor AC, Zhao KT, Packer MS, Gaudelli NM, Waterbury AL, Koblan LW, et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Science Advances. 2017;3(8):eaao4774. https://doi.org/10.1126/sciadv.aao4774.
Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature. 2017;551(7681):464–71. https://doi.org/10.1038/nature24644.
Lam DK, Feliciano PR, Arif A, Bohnuud T, Fernandez TP, Gehrke JM, et al. Improved cytosine base editors generated from TadA variants. Nat Biotechnol. 2023;41(5):686–97. https://doi.org/10.1038/s41587-022-01611-9.
Chen L, Park JE, Paa P, Rajakumar PD, Prekop HT, Chew YT, et al. Programmable C: G to G: C genome editing with CRISPR-Cas9-directed base excision repair proteins. Nat Commun. 2021;12(1):1384. https://doi.org/10.1038/s41467-021-21559-9.
Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016;533(7603):420–4. https://doi.org/10.1038/nature17946.
Neugebauer ME, Hsu A, Arbab M, Krasnow NA, McElroy AN, Pandey S, et al. Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. Nat Biotechnol. 2023;41(5):673–85. https://doi.org/10.1038/s41587-022-01533-6.
Shelake RM, Pramanik D, Kim J-Y. Improved Dual Base Editor Systems (iACBEs) for Simultaneous Conversion of Adenine and Cytosine in the Bacterium Escherichia coli. mBio. 2023;14(1):e02296–22. https://doi.org/10.1128/mbio.02296-22.
Zhao D, Li J, Li S, Xin X, Hu M, Price MA, et al. Glycosylase base editors enable C-to-A and C-to-G base changes. Nat Biotechnol. 2021;39(1):35–40. https://doi.org/10.1038/s41587-020-0592-2.
Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science. 2016;353(6305):aaf8729. https://doi.org/10.1126/science.aaf8729.
Lee M, Heo YB, Woo HM. Cytosine base editing in cyanobacteria by repressing archaic Type IV uracil-DNA glycosylase. Plant J. 2023;113(3):610–25. https://doi.org/10.1111/tpj.16074.
He Y, Zhou X, Chang C, Chen G, Liu W, Li G, et al. Protein language models-assisted optimization of a uracil-N-glycosylase variant enables programmable T-to-G and T-to-C base editing. Mol Cell. 2024;84(7):1257-70.e6. https://doi.org/10.1016/j.molcel.2024.01.021.
Tong H, Wang H, Wang X, Liu N, Li G, Wu D, et al. Development of deaminase-free T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase. Nat Commun. 2024;15(1):4897. https://doi.org/10.1038/s41467-024-49343-5.
Tong H, Wang X, Liu Y, Liu N, Li Y, Luo J, et al. Programmable A-to-Y base editing by fusing an adenine base editor with an N-methylpurine DNA glycosylase. Nat Biotechnol. 2023;41(8):1080–4. https://doi.org/10.1038/s41587-022-01595-6.
Niu T-C, Lin G-M, Xie L-R, Wang Z-Q, Xing W-Y, Zhang J-Y, et al. Expanding the Potential of CRISPR-Cpf1-Based Genome Editing Technology in the Cyanobacterium Anabaena PCC 7120. ACS Synth Biol. 2019;8(1):170–80. https://doi.org/10.1021/acssynbio.8b00437.
Sengupta A, Bandyopadhyay A, Sarkar D, Hendry JI, Schubert MG, Liu D, et al. Genome streamlining to improve performance of a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973. mBio. 2024;15(3):e03530–23. https://doi.org/10.1128/mbio.03530-23.
Sengupta A, Bandyopadhyay A, Schubert MG, Church GM, Pakrasi HB. Antenna Modification in a Fast-Growing Cyanobacterium Synechococcus elongatus UTEX 2973 Leads to Improved Efficiency and Carbon-Neutral Productivity. Microbiology Spectrum. 2023;11(4):e00500-e523. https://doi.org/10.1128/spectrum.00500-23.
Zheng Y, Su T, Qi Q. Microbial CRISPRi and CRISPRa Systems for Metabolic Engineering. Biotechnol Bioprocess Eng. 2019;24(4):579–91. https://doi.org/10.1007/s12257-019-0107-5.
Safari F, Zare K, Negahdaripour M, Barekati-Mowahed M, Ghasemi Y. CRISPR Cpf1 proteins: structure, function and implications for genome editing. Cell Biosci. 2019;9(1):36. https://doi.org/10.1186/s13578-019-0298-7.
Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat Protoc. 2013;8(11):2180–96. https://doi.org/10.1038/nprot.2013.132.
Kampmann M. CRISPRi and CRISPRa Screens in Mammalian Cells for Precision Biology and Medicine. ACS Chem Biol. 2018;13(2):406–16. https://doi.org/10.1021/acschembio.7b00657.
Huang C-H, Shen CR, Li H, Sung L-Y, Wu M-Y, Hu Y-C. CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942. Microbial Cell Factories. 2016;15(1):196. https://doi.org/10.1186/s12934-016-0595-3.
Lai MJ, Tsai JC, Lan EI. CRISPRi-enhanced direct photosynthetic conversion of carbon dioxide to succinic acid by metabolically engineered cyanobacteria. Biores Technol. 2022;366: 128131. https://doi.org/10.1016/j.biortech.2022.128131.
Santos M, Pacheco CC, Yao L, Hudson EP, Tamagnini P. CRISPRi as a Tool to Repress Multiple Copies of Extracellular Polymeric Substances (EPS)-Related Genes in the Cyanobacterium Synechocystis sp. PCC 6803. Life. 2021;11(11):1198. https://doi.org/10.3390/life11111198.
Zetsche B, Gootenberg Jonathan S, Abudayyeh Omar O, Slaymaker Ian M, Makarova Kira S, Essletzbichler P, et al. Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell. 2015;163(3):759–71. https://doi.org/10.1016/j.cell.2015.09.038.
Choi SY, Woo HM. CRISPRi-dCas12a: A dCas12a-Mediated CRISPR Interference for Repression of Multiple Genes and Metabolic Engineering in Cyanobacteria. ACS Synth Biol. 2020;9(9):2351–61. https://doi.org/10.1021/acssynbio.0c00091.
Knoot CJ, Biswas S, Pakrasi HB. Tunable Repression of Key Photosynthetic Processes Using Cas12a CRISPR Interference in the Fast-Growing Cyanobacterium Synechococcus sp. UTEX 2973. ACS Synthetic Biology. 2020;9(1):132–43. https://doi.org/10.1021/acssynbio.9b00417.
Liu D, Johnson VM, Pakrasi HB. A Reversibly Induced CRISPRi System Targeting Photosystem II in the Cyanobacterium Synechocystis sp. PCC 6803. ACS Synthetic Biology. 2020;9(6):1441–9. https://doi.org/10.1021/acssynbio.0c00106.
Yao L, Shabestary K, Björk SM, Asplund-Samuelsson J, Joensson HN, Jahn M, et al. Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes. Nat Commun. 2020;11(1):1666. https://doi.org/10.1038/s41467-020-15491-7.
Scholz I, Lott SC, Behler J, Gärtner K, Hagemann M, Hess WR. Divergent methylation of CRISPR repeats and cas genes in a subtype I-D CRISPR-Cas-system. BMC Microbiol. 2019;19(1):147. https://doi.org/10.1186/s12866-019-1526-3.
Funding
This research was supported by grants from the National Key Research and Development Program of China (Grant no. 2019YFA0904600), the National Natural Science Foundation of China (Grant nos. 32270091, 31972931 and 32070083).
Author information
Authors and Affiliations
Contributions
ZXD wrote the draft manuscript; LC and YW designed the manuscript; TS and WZ designed and revised the manuscript. All authors read and approved the final manuscript.
Corresponding authors
Ethics declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Dong, Z., Chen, L., Wang, Y. et al. Current advances in CRISPR-Cas-mediated gene editing and regulation in cyanobacteria. Blue Biotechnology 1, 9 (2024). https://doi.org/10.1186/s44315-024-00009-3
Received:
Accepted:
Published:
DOI: https://doi.org/10.1186/s44315-024-00009-3