Part:BBa_K3868098
pTargetS
pTargetS plasmid contains pj23119, sgRNA-1 and sgRNA-2 etc.Two different sgRNA expression frames were tandemly linked, allowing GGGGGGGG to be covered, resulting in a more diverse editing outcome.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 182
Illegal EcoRI site found at 552
Illegal XbaI site found at 188
Illegal XbaI site found at 558
Illegal SpeI site found at 73
Illegal SpeI site found at 443
Illegal PstI site found at 200
Illegal PstI site found at 570 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 182
Illegal EcoRI site found at 552
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 50
Illegal NheI site found at 377
Illegal NheI site found at 400
Illegal NheI site found at 420
Illegal SpeI site found at 73
Illegal SpeI site found at 443
Illegal PstI site found at 200
Illegal PstI site found at 570 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 182
Illegal EcoRI site found at 552
Illegal BglII site found at 212
Illegal BglII site found at 582
Illegal XhoI site found at 236
Illegal XhoI site found at 606 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 182
Illegal EcoRI site found at 552
Illegal XbaI site found at 188
Illegal XbaI site found at 558
Illegal SpeI site found at 73
Illegal SpeI site found at 443
Illegal PstI site found at 200
Illegal PstI site found at 570 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 182
Illegal EcoRI site found at 552
Illegal XbaI site found at 188
Illegal XbaI site found at 558
Illegal SpeI site found at 73
Illegal SpeI site found at 443
Illegal PstI site found at 200
Illegal PstI site found at 570 - 1000COMPATIBLE 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 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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