Target RNA single base editing system and method
An RNA editing and base editing technology, applied in microorganism-based methods, chemical instruments and methods, biochemical equipment and methods, etc., can solve the structure and molecular weight of Cas proteins, accommodate Cas protein-based base editors and Guide elements, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0161] Purification of SBDmmo-ADAR2 fusion protein
[0162] 1. Through gene synthesis, ADAR2DD (E488Q / T375G) was synthesized into the yeast expression vector pESC-URA, and SalI and BamHI restriction sites were added. The synthetic sequence is as follows (SEQ ID No: 1):
[0163]
[0164]
[0165]Among them, the bold part is the carrier sequence, the lowercase sequence is the SalI and BamHI restriction site, the italic part is the ADAR2DD (E488Q / T375G) sequence, and the underlined part is the histidine tag sequence.
[0166] The SBDmmo-ADAR2 fusion protein expression vector was constructed based on the above plasmids, and the amplification primer sequences were as follows:
[0167] SBDmmo-F: 5'-CCGctcgagATGATTAGCCCGGAAACCCT-3' (SEQ ID No: 2)
[0168] SBDmmo-R: 5'-CGCggatccGCTTCCACCCTCCTCCACGGAAATGCGGATCGCGGT-3' (SEQ ID No: 3)
[0169] 2. Heterologous expression of SBDmmo-ADAR2 fusion protein in Saccharomyces cerevisiae host BY4741. Pick the single clone carrying the exp...
Embodiment 2
[0177] RNA single base editing of green fluorescent protein in vitro using SBDmmo-ADAR2 fusion protein and gDNA
[0178] 1. Preparation of GFP (green fluorescent protein) mutant RNA substrate
[0179] The GFP gene was constructed on the pET28a Escherichia coli protein expression vector, and the GFP DNA sequence was point-mutated using mutation primers, so that one of the Gs was mutated into A. The sequence of the mutation primers was as follows:
[0180] GFP-Mut-F: 5'-TGTTCCATAGCCAACACTTGTCACT-3' (SEQ ID No: 5)
[0181] GFP-Mut-R: 5'-GTGTTGGCTATGGAACAGGCAGCTT-3' (SEQ ID No: 6)
[0182] After mutation, a stop codon is introduced inside GFP, so that GFP cannot be completely expressed in the expression host, and the host does not emit green fluorescence.
[0183] Use the RNA transcription kit to obtain the mutated GFP mutant RNA substrate, add DNaseI to remove the residual DNA in the system, and use PCR to verify that there is no DNA residue.
[0184] 2. gDNA (guid DNA) prepar...
Embodiment 3
[0204] RNA single-base editing of green fluorescent protein in vitro using SBDmmo-ADAR2 fusion protein and sulfur-modified gRNA
[0205] 1. The GFP (green fluorescent protein) mutant RNA substrate is consistent with that in Example 2.
[0206] 2. gRNA (guid RNA) preparation
[0207] Design a single-stranded sulfur-modified gRNA at the GFP mutation site. Referring to the results in Example 1, two single-stranded sulfur-modified gRNAs, Grna-13 and gRNA-7S, were designed. The gRNA is complementary to the GFP sequence as a whole, but at the mutation site A C-A mismatch is formed at the point, the sulfur modification site in the gRNA is located at the 3' end of the mutation site, and a gRNA-0 without sulfur modification was designed as a negative control, the sequence is as follows:
[0208]gRNA-13:5'-ACAAGUGUUGGCCAUGGAACAGGCAG*CUUGCCGGUAGUGC-3' (SEQ ID No: 15)
[0209] gRNA-7S:5'-ACAAGUGUUGGCCAUGGAACAGGCAG*C*U*U*G*C*C*GGUAGUGC-3' (SEQ IDNo: 16)
[0210] gRNA-0: 5'-ACAAGUGUUGGCC...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More