1. CRISPR-Based Technologies for Metabolic Engineering in Cyanobacteria
Juliane Behler, Dhanya Vijay, Wolfgang R. Hess, M. Kalim Akhtar 
Trends in Biotechnology 
Volume 36, Issue 10, Pages (October 2018)
DOI: /j.tibtech
Copyright © 2018 The Authors Terms and Conditions

2. Figure 1
Metabolic Engineering of Cyanobacteria for Succinate Production Using CRISPR-Based Approaches. (A) Metabolic scheme for the production of succinate. Under nitrogen-starvation conditions, glycogen serves as a major carbon sink in cyanobacteria. Removal of glgC eliminates the main route toward glycogen synthesis and provides additional glyceraldehyde-3-phosphate (G3P) for succinate production, facilitated by the expression of ppc and gltA. Only key enzymes are shown and marked in red. Upward arrows indicate gene knock-in, downward arrows indicate repression. (B) Application of CRISPR/Cas9 for succinate production. A plasmid-based CRISPR/Cas9 system was used to knockout glgC, and knock-in ppc and gltA for succinate production in Synechococcus elongatus PCC 7942 [24]. Cas9, tracrRNA, and glg C-targeting crRNA were expressed in trans. The second plasmid encoded both gltA and ppc for simultaneous knock-in and an appropriate selection marker for gentamicin resistance (GmR). Genes subjected to knock-in mutations were flanked by corresponding homology regions (blue and orange sections of plasmid) serving as donor DNA for the simultaneous knockout/knock-in approach. Cas9 introduces a double-strand break in a PAM sequence 5′-NGG-3′-dependent manner and the donor DNA with homology regions serves as a template for homology-directed repair. (C) Application of CRISPRi/dCas9 for succinate production. Native glgC, sdhA, and sdhB genes were used as target genes for repression in S. elongatus PCC 7942 [37]. The dCas9 and sgRNA cassettes targeting these genes were first integrated into the wild-type chromosome at the NSI and NSII neutral sites, respectively. Upon induction of dCas9, the sgRNA directs dCas9 to its place of transcriptional inhibition in a 5′-NGG-3′ PAM-dependent manner. This leads to sterical blocking (red flash) of the RNA polymerase during the transcription process resulting in the dissociation of the enzyme and transcriptional inhibition of the targeted gene. Abbreviations: Cas, CRISPR associated; Cit, citrate; CoA, coenzyme A; CRISPR, clustered regularly interspaced short palindromic repeats; crRNA, CRISPR RNA; FUM, fumarate; dCas9, dead Cas9; G1P, glucose-1-phosphate; G3P, glyceraldehyde-3-phosphate; glgC, glucose-1-phosphate adenylyltransferase; gltA, citrate synthase; OOA, oxaloacetate; PAM, protospacer adjacent motif; PEP, phosphoenolpyruvate; ppc, phosphoenolpyruvate carboxylase; PYR, pyruvate; PCC, Pasteur Culture Collection; SDH, succinate dehydrogenase; sgRNA, single-guide RNA; sdhA and sdhB, encode subunits of succinate dehydrogenase; tracrRNA, trans-activating CRISPR RNA.
Trends in Biotechnology  , DOI: ( /j.tibtech )
Copyright © 2018 The Authors Terms and Conditions

3. Figure I
Illustration of Unicellular (Left) and Filamentous (Right) Cyanobacteria.
Trends in Biotechnology  , DOI: ( /j.tibtech )
Copyright © 2018 The Authors Terms and Conditions

4. Figure I
CRISPR Effectors Used for Cyanobacterial Engineering. (A) Arrangement of loci and domain structures of the major CRISPR effector proteins. While the CRISPR/Cas9 system relies on an additional trans-activating crRNA (tracrRNA) for CRISPR processing, the Francisella novicida CRISPR/Cas12a system and the Leptotrichia shahii CRISPR/Cas13a system lack tracrRNA. The Cas proteins, along with the CRISPR arrays and any additional RNA molecules, constitute the core functional unit of the CRISPR/Cas bacterial immune system. (B) Applying the CRISPR/Cas12a system for editing the genome of cyanobacteria. A markerless nblA deletion mutant was obtained using CRISPR/Cas12a in Synechococcus elongatus UTEX 2973 [28]. Cas12a harbors one RNA cleavage domain and a split RuvC motif (RuvC I, RuvC II, and RuvC III) for DNA cleavage, as elucidated by Fonfara and colleagues [31]. Thus, Cas12a maintains a dual function by mediating both the maturation of the nblA-targeting crRNA and the introduction of a 5-bp staggered double-stranded break (DSB) within the chromosome located downstream of a PAM site (5′-YTN-3′). The pSL2680-based vector (Addgene, plasmid #85581) expresses both Cas12a and the nblA-targeting crRNA and contains 1-kb homology regions complementing the target gene locus to be edited. Cas12a and the nblA crRNA were expressed under the control of the inducible lac and the constitutive J23119 promoter, respectively. The nblA crRNA-encoding array contains the F. novicida leader sequence (L) followed by direct repeat (DR), spacer (S) sequences, and the F. novicida terminator (T). After the introduction of the DSB, the homologous regions within the donor DNA serve as templates for the homology-directed repair mechanisms and cells can be cured of the editing plasmid. Since NblA is involved in the degradation of the phycobilisomes during nitrogen starvation, nblA knockout mutants show a nonbleaching phenotype due to the impaired ability to degrade phycobilisomes, whereas wild-type cells bleach distinctly [28]. Abbreviations: crRNA, CRISPR RNA; R, repeat; S, spacer.
Trends in Biotechnology  , DOI: ( /j.tibtech )
Copyright © 2018 The Authors Terms and Conditions