CRISPR/Cas9-mediated method for splicing large fragments of DNA
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A DNA sequence and nucleic acid construct technology, applied in the field of large fragment DNA splicing, can solve the problems of difficult operation and low efficiency
Active Publication Date: 2021-12-14
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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Although the cost of DNA synthesis continues to decrease with the advancement of technology, the assembly of very large fragments of DNA or even complete genomes is still relatively inefficient due to the difficulty in operating large fragments of DNA.
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Embodiment 1
[0066] Example 1 : Splicing of two large fragments of DNA
[0067] In this example, the pSP5 and pTP3-U plasmids constructed in Saccharomyces cerevisiae VL6-48 in our laboratory were selected (Table 2). pSP5 contains 116643bp of donor DNA, named SP5; pTP3-U contains 184475bp of donor DNA, named TP3. Both SP5 and TP3 sequences are derived from Escherichia coli, which are large plasmids obtained by amplifying certain essential genes of Escherichia coli by PCR and splicing two or three levels in yeast. The right end of SP5 and the left end of TP3 have the same 491bp source sequence.
[0068] Table 2: Plasmid pSP5, pTP3-U information
[0069] Donor plasmid filter marker Donor DNA name Insert size (bp) pSP5 HIS3 SP5 116643
[0070] pTP3-U URA3 TP3 184475
[0071] The Cas9 expression plasmid pMetcas9 ( image 3 , SEQ ID NO: 1) was transferred into the yeast cell VL6-48 containing the pSP5 plasmid. Protoplast fusion of VL6-48 / p...
Embodiment 2
[0120] Example 2 : Splicing of three large fragments of DNA
[0121] In this example, the pTP1, pTP2 and pTP3-L plasmids constructed in Saccharomyces cerevisiae VL6-48 in our laboratory were selected (Table 4).
[0122] Table 4: Plasmid pTP1, pTP2, pTP3-L information
[0124] pTP1 contains 177147bp of donor DNA, named TP1; pTP2 contains 297952bp of donor DNA, named TP2; pTP3-L contains 184475bp of donor DNA, named TP3. The sequences of TP1, TP2 and TP3 are all derived from Escherichia coli, and their construction methods are similar to those of pSP5 and pTP3-U in Example 1. There is a 385bp homologous sequence between the right end of TP1 and the left end of TP2, and a 491bp homologous sequence between the right end of TP2 and the left end of TP3. The Cas9 expression plasmid pMetcas9 (see image 3 and ...
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Abstract
The invention discloses a CRISPR / Cas9-mediated large fragment DNA splicing method. Specifically, the present invention provides a nucleic acid construct capable of constitutively expressing Cas9 in yeast, which contains a yeast Tef1 promoter operably linked to the cas9 gene, an origin of replication from pBR322 and a selection marker and The replication region derived from CEN6ARS4 and its selection marker, the nucleic acid construct has single-copy replication in yeast and high-copy replication in Escherichia coli. The invention also provides a nucleic acid construct capable of constitutively expressing sgRNA in yeast, a nucleic acid construct used as an acceptor vector, and a DNA splicing method. Using the present invention, two or more large DNA fragments to be spliced can be successfully spliced into large plasmids in yeast at one time, eliminating the need for in vitro enzyme digestion recovery and genetic transformation of low-efficiency large fragment DNA, which is more convenient than traditional methods efficient.
Description
technical field [0001] The invention relates to the fields of microbial synthetic biology, genome engineering and molecular biology, in particular to a CRISPR / Cas9-mediated method for splicing large fragments of DNA. Background technique [0002] Synthetic biology is an emerging field of research. In 2010, American scientist Venter and his research team reported the world's first artificial life [Gibson, D.G, etc., Creation of a bacterial cell controlled by a chemically synthesized genome, Science, 2010, 329(5987): p.52-6], This world-renowned research achievement has made synthetic biology a research hotspot in modern life sciences. The development of DNA synthesis, large fragment splicing and genome transplantation technology is an important technical basis in synthetic biology research. DNA splicing developed by Venter and his research team [Gibson, D.G. et al., Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome, Science, 2008, 319(5867...
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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12N15/70C12N1/19C12N1/21
Inventor 覃重军周见庭吴荣海薛小莉
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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