AAV virus-based gene editing expression cassette
A technology of expressing cassettes and viruses, which is applied in the field of gene editing, and can solve problems such as limited scope of use and small carrying capacity
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Embodiment 1
[0085] Example 1 Construction of pX601(EF1α-tRNA) vector
[0086] 1.1 Construction of pX601(tRNA) vector
[0087] The present invention uses the Gln tRNA coding sequence (SEQ ID NO: 7) as the promoter of the promoter-sgRNA unit, and the coding sequence is synthesized by Shanghai Sangong Company as a tRNA-SP plasmid, which is used as a template (10ng / system), to Primers:
[0088] PA-tRNA-F:
[0089] 5′-AGGCATGCTGGGGAGGTACCGGTTCCATGGTGTAATGGTT-3′ and
[0090] tRNA(SpCas)(VB)-R:
[0091] 5′-ACAGGTCTTCTCGAAGACCCAGGTTCCCACCGAGATTTGAA-3′
[0092] Perform PCR amplification;
[0093] At the same time, use the pX601 plasmid (purchased from Addgene, plasmid number 61591) as a template (10ng / system), and use the primers:
[0094] tRNA(SpCas)-F:
[0095] 5′-CTGGGTCTTCGAGAAGACCT-3′ and
[0096] Scaf-ITR-R:
[0097] 5′-CTAGGGGTTCCTGCGGCCGCAAAAAAATCTCGCCAACAAGTTG-3′
[0098] PCR amplification was carried out with Q5 DNA polymerase respectively. After the amplification, 1 μL of Dpn ...
Embodiment 2
[0116] Embodiment 2 constructs the recombinant vector containing spacer sequence
[0117] 2.1 Design and construction of scaffold-promoter recombination vector containing spacer sequence
[0118] Design primers as required as shown in Table 1:
[0119] Table 1
[0120]
[0121]
[0122] Use pX601(EF1α-tRNA) plasmid as template (10ng / system), with primers: scaf-F / scaf-R, scaf-F / scaf-10-R, scaf-tRNA-F / tRNA-R, scaf-10 - PCR amplification of tRNA-F / tRNA-R, scaf-20-tRNA-F / tRNA-R, scaf-40-tRNA-F / tRNA-R, adding 0bp, 10bp, 20bp and Scaffold-tRNA plasmid with 40 bp spacer.
[0123] At the same time, using the pX601 plasmid as a template (10ng / system), primers: scaf-U6-F / U6-R, scaf-10-U6-F / U6-R, scaf-20-U6-F / U6-R, Scaf-40-U6-F / U6-R was amplified by PCR with Q5 DNA polymerase, respectively, to obtain scaffold-U6 plasmids in which 0bp, 10bp, 20bp and 40bp spacer sequences were sequentially added between the scaffold and the U6 promoter. After the amplification, the plasmid temp...
Embodiment 3
[0126] Example 3 Construction of sgRNA tandem recombination vector targeting multiple targets
[0127] In the present invention, 4 sgRNA sites of 3 mouse endogenous genes are used in order: mMSTN-sgRNA1, mMSTN-sgRNA2, mTyr-sgRNA3, mRosa26-sgRNA2, and pX601 is used as the carrier skeleton to construct the sgRNA tandem recombination vector .
[0128] Using scaffold-U6 or scaffold-tRNA as template, design primers for PCR amplification, wherein, sg1-F primer (5'-3') includes 20bp of U6 or tRNA 3' end sequence, plus the guide of the first sgRNA Sequence (approximately 22bp), plus 18bp of scaffold 5' end sequence; sgN-R primer (5'-3') includes 20bp reverse scaffold 5' end sequence, plus reverse Nth sgRNA guide Sequence (about 22bp), plus 18bp reverse U6 or tRNA 3' end sequence; other sgRNA primer sequences have specific guide sequence, forward primer also has 18bp scaffold 5' end sequence, reverse primer Then it has 18bp of U6 or tRNA3' end sequence. The primer sequences are show...
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