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Our project tested the efficiency of our accelerators using three plasmid architectures: single plasmid system
(Cas12a, Argonaute) and a double plasmid system (Cas12a).
These designs were inspired by "A toolbox to engineer the highly productive cyanobacteria PCC 11901”
We assembled the plasmids using the MoClo Cyanogate Assembly kit [1][2][3], with the double plasmid system drawing inspiration
-- a paper from Alistair McCormick’s lab. [4]
Argonaute System
Research regarding Argonaute proteins has primarily focused on their roles in eukaryotic organisms and their involvement in RNA
interference systems used for post-transcriptional gene silencing. [9] As research in this area progressed,
Argonaute proteins were discovered in various organisms, including prokaryotes; these prokaryotic Argonaute proteins are commonly referred to as pAgos.
The Ago proteins are nucleic acid-guided endonucleases, and can be guided with RNA or DNA to cleave either RNA or DNA, respectively. Their main function is to
protect the cell against exogenous DNA by silencing certain gene expressions through cleavage. The Ago protein and a small DNA guide (smDNA),
complementary to the target form a complex that targets foreign DNA and cleaves the polynucleotide chain.
The Ago complex is well characterized and it comprises 4 main parts: PIWI, MID, PAZ, and N domains. [10] The PIWI domain is the
active site responsible for cleaving the target nucleic acid strand after the smDNA has paired with the target. The MID domain binds to the 5' phosphorylated end of a
16-18 nucleotide-long single-stranded smDNA guide, securing it in place for the target search. The PAZ domain plays a critical role in locating the complementary sequence
on the target strand, holding onto the 3' end of the guide to ensure proper alignment for the PIWI domain to execute cleavage. Once the target strand is cleaved, the N
domain facilitates the release of the cleaved strand, allowing the protein complex to reset for another round of target binding.
When it comes to genomic editing and regulation, RecBCD and RecA play integral roles with CbAgo. RecBCD binds to the single strand end and digests the DNA sequence
until a Chi site is found. It is important to note that RecBCD acts as its own helicase and nuclease. Upon recognition of a Chi Site (sequence: GCGATCGC), RecBCD
stops digesting DNA; from here RecA binds to single stranded DNA and recognizes homologous double stranded DNA producing a RecA filament
[10] This filament promotes homologous DNA repair after RecBCD digests. In regards to our strain of UTEX 3154,
even though RecBCD isn’t present, homologous recombination still occurs; this led us to the hypothesis that there may be native enzyme complex that may
possess identical or similar function as RecBCD.
We have a replicative plasmid containing our Argonaute protein and 1200 base pairs of a homologous region to the genome called the AquI locus. [4] The replicative nature of the plasmid will allow many Argonaute proteins to be present. Regarding our Argonaute protein, we use a pBAD promoter which is inducible based on the presence of Arabinose. In other words, the Argonaute protein will only be expressed when Arabinose is present.[5] In terms of our locus region, we chose AquI as it is a restriction enzyme site, and targeting the gene for a knockout will not affect our strain. By splitting them into three 400bp regions, we made our experiments more customizable when introducing Chi Sites and Restriction Enzyme Sites to test their significance in guide DNA loading of Argonaute. [6]
We have a suicide plasmid containing the homology arms, green fluorescent protein (GFP), and Spectinomycin resistance marker.
The suicide nature of the plasmid provides the benefit of killing the plasmid after having the GFP and Spectinomycin integrated into the genome.
The homology arms will act as the adhesive part of a bandaid for the genome while GFP and Spectinomycin resistance will act as the pad of the bandaid for the genome.
By homologous recombination, we can introduce our inserts (GFP and Spec) into the strain’s genome.
Learn more about specific parts used in the Argonaute system >>
Single Plasmid System
Our initial approach to speeding up our process involved employing the CRISPR system. Research indicates that Cpf1, a CRISPR-associated protein, is less toxic than Cas9, leading us to compare their toxicity. [11] This was done by monitoring the growth of cells containing both CRISPR systems, with an empty vector serving as a control. Cpf1 demonstrated a cell-death of 50%, while Cas9 containing cells exhibited no cell-growth except for four colonies. The study further demonstrates data that shows the effectiveness of Cas12a in Synechococcus 2973 and Anabaena 7120, demonstrating deletions, knock-ins, and direct gene replacement. In our project, we plan on using Cas12a as an established system for the genetic modification of UTEX 2973; the accelerators we design in conjunction may improve the reliability of stable integrants. We primarily chose Cas12a for its lower toxicity to the cells, and it came with additional benefits making it more efficient for genetic engineering in cyanobacteria. The dual nuclease operates pre-crRNA processions and DNA cleaving, providing much of the capabilities of Cas9. It also works better for the cutting of DNA, as it cuts with a 5 base pair offset that preserves the PAM site. For repeated cloning, this would be advantageous as we modify multiple copies of the genome present within cyanobacteria. Cas12a does not require the insertion of a tracrRNA to mediate the activation of the protein, which vastly simplifies the system and allows efficient genetic engineering within the polyploid system. The system also confers less metabolic strain on the host, as its protein is roughly 20% smaller and it requires less RNA to operate in comparison to Cas9.
This Cpf1 system relies on combining our payload, which like our Argonaute system also contains GFP and spectinomycin resistance,
and our Cpf1 accelerator into a single plasmid into a RSF1010 based replicative backbone.[7]
This Cpf1 accelerator system relies on the inducible pBAD promoter, while our sgRNA cassette is promoted separately by the J23119 promoter.
Our system will also be targeting the AquI locus, which is done using our sgRNA cassette which will express seven separate guide RNAs specific to locus.
Like the Argonaute system, the goal is to induce homologous recombination with the payload section of our plasmid through double strand breaks in the genome.
By using a single plasmid, we will be able to use the natural transformation ability of PCC 11901 to introduce our plasmid system rather than relying on
conjugation protocols. [4]
Learn more about specific parts used in the Single Plasmid system >>
Double Plasmid System
The inspiration for the double plasmid system came from a paper written by Alistair McCormick’s Lab: “A toolbox to engineer the highly productive cyanobacteria PCC 11901”.[4] This method was selected as a baseline for our argonaute experiments because of its efficiency of establishing fully segregated integrants. The system relies on a two step homologous recombination process to achieve full markerless integration. First, the editing RSF1010 plasmid is introduced into our chosen strain through conjugation. This plasmid, which has a high copy number, generates multiple copies within the cell while also carrying our repressed Cas12a cassette. To prevent transient expression of Cas12a, its expression is tightly regulated by a highly repressed promoter (phlF). Another promoter (pJ23101) controls the expression of the repressor phlF, which is derepressed by DAPG to enable Cas12a expression.
While our system is inspired by Victoria et al., some of the components are slightly different. Our Cas12a gene block was derived from the pSL2680 plasmid,
which we codon-optimized and analyzed using our program. Additionally, the cassette for Gentamicin resistance was also processed with STEALTH. [8]
The RSF1010 editing plasmid is assembled via inverse PCR of the pC1.509 construct which produces a product similar to the level T backbone acceptors in the
CyanoGate kit. Our Cas12a gene will be assembled into the RSF1010 backbone via PCR, with primers that include the necessary MoClo overhangs.
The primers are also designed to remove any downstream nonsense base pairs during digestion. This setup bypasses the need for intermediate Golden Gate Assembly
reactions, since our Cas12a gene block will be directly assembled into the RSF1010 backbone.
Learn more about specific parts used in the Double Plasmid system >>
References
[1] A modular cloning system for standardized assembly of multigene constructs. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. PLOS ONE . 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765. PubMed PMID 21364738.
[2] Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system. Werner S, Engler C, Weber E, Gruetzner R, Marillonnet S. Bioeng Bugs. 2012 Jan 1;3(1):38-43. doi: 10.1371/journal.pone.0016765. PubMed PMID 22126803.
[3] CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax. Vasudevan R, Gale GAR, Schiavon AA, Puzorjov A, Malin J, Gillespie MD, Vavitsas K, Zulkower V, Wang B, Howe CJ, Lea-Smith DJ, McCormick AJ. Plant Physiology.2019 May;180(1):39-55. doi: 10.1104/pp.18.01401. PubMed PMID: 30819783.
[4] A. J. Victoria et al., “A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901,” PLANT PHYSIOLOGY, May 2024, doi: https://doi.org/10.1093/plphys/kiae261.
[5]“Tightly Controlled Bacterial Protein Expression pBAD Expression System The pBAD system offers: • Tightly regulated expression • Dose-dependent induction • High protein yields • Simplified detection and purification of expressed protein.” Available: https://assets.thermofisher.com/TFS-Assets/LSG/brochures/710_01619_pBAD_bro.pdf
[6] S. Huang, K. Wang, and S. L. Mayo, “Genome manipulation by guide-directed Argonaute cleavage,” Nucleic Acids Research, vol. 51, no. 8, pp. 4078–4085, Mar. 2023, doi: https://doi.org/10.1093/nar/gkad188.
[7] S. Baldanta, G. Guevara, and J. M. Navarro-Llorens, “SEVA-Cpf1, a CRISPR-Cas12a vector for genome editing in cyanobacteria,” Microbial Cell Factories, vol. 21, no. 1, May 2022, doi: https://doi.org/10.1186/s12934-022-01830-4.
[8] S. Hu, S. Giacopazzi, R. Modlin, K. Karplus, D. L. Bernick, and K. M. Ottemann, “Altering under-represented DNA sequences elevates bacterial transformation efficiency,” mBio, vol. 14, no. 6, Oct. 2023, doi: https://doi.org/10.1128/mbio.02105-23.
[9] J. Wu, J. Yang, W. C. Cho, and Y. Zheng, “Argonaute proteins: Structural features, functions and emerging roles,” Journal of Advanced Research, vol. 24, pp. 317–324, Apr. 2020, doi: https://doi.org/10.1016/j.jare.2020.04.017.
[10] B. A. Graver, N. Chakravarty, and K. V. Solomon, “Prokaryotic Argonautes for in vivo biotechnology and molecular diagnostics,” Trends in Biotechnology, vol. 42, no. 1, pp. 61–73, Jan. 2024, doi: https://doi.org/10.1016/j.tibtech.2023.06.010.
[11] J. Ungerer and H. B. Pakrasi, “Cpf1 Is A Versatile Tool for CRISPR Genome Editing Across Diverse Species of Cyanobacteria,” Scientific Reports, vol. 6, no. 1, Dec. 2016, doi: https://doi.org/10.1038/srep39681.