Part:BBa_K3904402

pta-specific sgRNA

Introduction

Vilnius-Lithuania iGEM 2021 project AmeByelooks at amebiasis holistically and comprehensively, therefore target E. histolytica from several angles: prevention and diagnostics. Our team's preventive solution consists of probiotics engineered to produce naringenin - an antiprotozoal compound. Two strains of genetically modified microorganisms were chosen as the main chassis - world-renowned Lactobacillus casei BL23 (Lactobacillus paracasei) and Escherichia coli Nissle 1917. Furthermore, the team made specific gene deletions to enhance naringenin production and adapted a novel toxin-antitoxin system to prevent GMO spreads into the environment. The diagnostic part includes a rapid, point of care, user-friendly diagnostic test identifying extraintestinal amebiasis. The main components of this test are aptamers specific to the E. histolytica secreted proteins. These single-stranded DNA sequences fold into tertiary structures for particular fit with target proteins.

Usage and Biology

CRISPR-Cas9 is a versatile genome-editing technique. In our approach to editing E. coli Nissle 1917 genome, we have used two plasmid based system enabling to combine of Lambda Red recombination and CRISPR-Cas9 as counterselection tools encoded in pCas and pTarget.

Mechanism of genome editing

pCas plasmid is used for Cas9, Lambda Red system expression, and plasmid curing of pTarget. Cas9 - the RNA-guided endonuclease - is expressed constitutively, while the expression of Lambda Red genes (Gam, Exo, Beta) is under the control of arabinose inducible promoter araBp. pTarget plasmid caries constitutively expressed single-guide RNA (sgRNA). This RNA molecule, as and in nature, is composed of 17-20 nt length guide RNA (gRNA) sequence complementary to the targeted DNA adjacent to the protospacer adjacent motif (PAM) present at the 3' end, and the scaffold for the Cas9 nuclease binding to sgRNA and forming the ribonucleoprotein complex (1). Although in nature sgRNA exists as two separate RNA molecules, in laboratory experiments they are usually combined into one single-guide RNA (sgRNA) obviating additional maturation steps. As both pCas and pTarget plasmids are in a cell, Cas9 nuclease and sgRNA are able to form ribonucleoprotein complex, scan DNA for PAM sequences and perform a double-strand break in a part of the DNA which is complementary to the gRNA and adjacent to the PAM sequence - NGG. However, if arabinose has been added to the cell culture and a double-stranded DNA repair template is present in the cell, the Lambda Red system performs homology directed repair (HDR). If this process is unsuccessful, the Cas9-sgRNA complex will cause a double-strand break and will cause cell death (2). This is employed as a counter selection in order to avoid the additional antibiotic as selection marker usage.

Table 1. sgRNA collection for E. coli Nissle 1917 genome editing.

ackA-specific sgRNA	BBa_K3904401
pta-specific sgRNA	BBa_K3904402
colicin-specific sgRNA	BBa_K3904405
nupG-specific sgRNA	BBa_K3904426

Table 2. Recombination templates collection for E. coli Nissle 1917 knockouts creation.

Recombination template for ackA knockout	BBa_K3904406
Recombination template for pta knockout	BBa_K3904407

pta gene knockout importance to AmeBye project

This RNA sequence is designed to target the CRISPR-Cas9 system to a phosphate acetyltransferase (pta) coding gene, which is one of the ackA-pta operon. pta is responsible for the catalyzation of reversible interconversion of acetyl-CoA and acetyl phosphate (3). Disruption of this genomic region is known to limit the acetate formation from acetyl-CoA, increasing the cellular concentration of acetyl-CoA up to 16% (4). Increased concentration of this molecule theoretically should result in enhanced malonyl-CoA formation and consequently more effective naringenin synthesis since the amount of malonyl-CoA available in the cell is the limiting step of naringenin production (5).

Genome editing efficiency

pta knockout generation with this sgRNA have achieved 60 % (fig. 1) and 80 % (fig. 2) efficiency .

Fig. 1. Restriction of cPCR product representing pta knockout generation. pta gene have been amplified from genomic DNA and restricted by BcuI. 2161 bp fragments represent wild type genotype, 1797 bp and 364 bp - knockouts. 1 - wild type (negative control), 2 - pta knockout (1), 3 - pta knockout (2), 4 - pta knockout (3), 5 - pta knockout (4), 6 - pta knockout (5), 7 - pta knockout (6), 8 - pta knockout (7), 9 - pta knockout (8), 10 - pta knockout (9), 11 - pta knockout (10).

Fig. 2. Restriction of cPCR product representing ackA-pta double knockout generation. pta gene have been amplified from genomic verified ackA knockout DNA and restricted by BcuI. 2161 bp fragments represent wild type genotype, 1797 bp and 364 bp - knockouts. 1 - wild type (negative control), 2 - no DNA added, 3 - ackA-pta knockout (1), 4 - ackA-pta knockout (2), 5 - ackA-pta knockout (3), 6 - ackA-pta knockout (4), 7 - ackA-pta knockout (5), 8 - ackA-pta knockout (6), 9 - ackA-pta knockout (7), 10 - ackA-pta knockout (8), 11 - ackA-pta knockout (9), 12 - ackA-pta knockout (10).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Doudna, J. A., & Charpentier, E. (2014). The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213).
2. Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., & Yang, S. (2015). Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Applied and environmental microbiology, 81(7), 2506-2514.
3. Dittrich, C. R., Bennett, G. N., & San, K. Y. (2005). Characterization of the acetate-producing pathways in Escherichia coli. Biotechnology progress, 21(4), 1062–1067.
4. Ku, J. T., Chen, A. Y., & Lan, E. I. (2020). Metabolic engineering design strategies for increasing acetyl-CoA flux. Metabolites, 10(4), 166.
5. Wu, J., Du, G., Chen, J., & Zhou, J. (2015). Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli. Scientific reports, 5(1), 1-14.