Part:BBa_K3868097

pCBE

The pCBE plasmid contains the lambda operator, cytidine deaminase, Uracil DNA glycosylase inhibitor and LVA degradation labels. The CBE / sgRNA complex can bind to the double-stranded DNA to form an R-loop in a sgRNA and PAM-dependent manner. CDA catalyzes the deamination of cytosines located at the top (non-complementary) strand within 15–20 bases upstream from PAM, which results in C-to-T mutagenesis.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1572
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1572
  Illegal NheI site found at 1331
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1572
  Illegal BglII site found at 5013
  Illegal BamHI site found at 3610
  Illegal XhoI site found at 4616
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1572
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1572
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

        The dual plasmids system of CBE was designed, built and tested. The pCBE plasmid contains the lambda operator, cytidine deaminase, Uracil DNA glycosylase inhibitor and LVA degradation labels. Based on the pCas, the pCBE (BBa_K3868097) was successfully constructed (Fig. 3A). To extend the editing range, two different sgRNA expression frames were tandemly linked, allowing GGGGGGGG to be covered, resulting in a more diverse editing outcome. Based on the pTarget, the pTargetS plasmid (BBa_K3868098) was successfully constructed (Fig. 3B), and the sequences of sgRNA1 and sgRNA2 was showed in Fig. 3C. The CBE / sgRNA complex can bind to the double-stranded DNA to form an R-loop in a sgRNA and PAM-dependent manner. CDA catalyzes the deamination of cytosines located at the top (non-complementary) strand within 15–20 bases upstream from PAM, which results in C-to-T mutagenesis.

Fig 3. A and B. The dual plasmid system was designed and used for CBE system. C. A schematic model for CBE.

Results

         During construction of the library, 90 single colonies were randomly selected for sequencing. It was found that 48 variants with different RBS sequence were identified from 90 samples, with an editing efficiency of only 53%. However, it is noteworthy that the transformants were grown on the solid medium for longer time, the reproducibility of the results gradually increased, and majority of variant RBS sequences became GAAAAAAG (Fig.4), probably due to the continuous base editing in the transformants. The above results show that although CBE possesses the advantages of simplicity and rapidity, editing results and efficiency applied in BL21 (DE3) are not sufficiently stable.

Fig 3. A and B. The dual plasmid system was designed and used for CBE system. C. A schematic model for CBE.

Results

         During construction of the library, 90 single colonies were randomly selected for sequencing. It was found that 48 variants with different RBS sequence were identified from 90 samples, with an editing efficiency of only 53%. However, it is noteworthy that the transformants were grown on the solid medium for longer time, the reproducibility of the results gradually increased, and majority of variant RBS sequences became GAAAAAAG (Fig.4), probably due to the continuous base editing in the transformants. The above results show that although CBE possesses the advantages of simplicity and rapidity, editing results and efficiency applied in BL21 (DE3) are not sufficiently stable.

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Fig 4. Schematic representation of the changes in G and A abundance of the RBS variant sequences of T7 RNAP obtained from CBEs experiments.

Reference

1. Gong G, Zhang Y, Wang Z, Liu L, Shi S, Siewers V, Yuan Q, Nielsen J, Zhang X, Liu Z. GTR 2.0: GRNA-tRNA array and Cas9-ng based genome disruption and single-nucleotide conversion in Saccharomyces cerevisiae. ACS synthetic biology. 2021; 10: 1328–1337.

2. Zhao D, Li J, Li S, Xin X, Hu M, Price MA, Rosser SJ, Bi C, Zhang X. Glycosylase base editors enable C-to-A and C-to-G base changes. Nature Biotechnology. 2021; 39: 35–40.

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Fig 4. Schematic representation of the changes in G and A abundance of the RBS variant sequences of T7 RNAP obtained from CBEs experiments.

Reference

1. Gong G, Zhang Y, Wang Z, Liu L, Shi S, Siewers V, Yuan Q, Nielsen J, Zhang X, Liu Z. GTR 2.0: GRNA-tRNA array and Cas9-ng based genome disruption and single-nucleotide conversion in Saccharomyces cerevisiae. ACS synthetic biology. 2021; 10: 1328–1337.

2. Zhao D, Li J, Li S, Xin X, Hu M, Price MA, Rosser SJ, Bi C, Zhang X. Glycosylase base editors enable C-to-A and C-to-G base changes. Nature Biotechnology. 2021; 39: 35–40.