CRISPR/Cas9-mediated large DNA fragment assembling method
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A technology for nucleic acid constructs and yeast, applied in recombinant DNA technology, fungi, bacteria, etc., can solve problems such as difficult operation and low efficiency
Active Publication Date: 2017-03-08
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 fra...
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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 DNA fragment assembling method. Specifically, the invention provides a nucleic acid construct capable of carrying out constitutive expression of Cas9 in yeast; the nucleic acid construct contains a yeast Tef1 promoter operationally connected with a Cas9 gene, a replication origin from pBR322 and a screening mark thereof, and a replication region from CEN6ARS4 and a screening mark thereof; and the nucleic acid construct is in the form of single-copy replication in the yeast and in the form of high-copy replication in Escherichia coli. The invention also provides a nucleic acid construct capable of carrying out constitutive expression of sgRNA in yeast, a nucleic acid construct used as a receptor vector and a DNA assembling method. According to the invention, two or more to-be-assembled large DNA fragments are successfully assembled in microzyme to form a large plasmid in one shot; so low-efficiency in-vitro digestion recovery, genetic transformation and the like of large DNA fragments are avoided, and the method provided by the invention is more convenient and efficient compared with traditional methods.
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 achemically 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 in vitro [Gibson, D.G. et al., Complete chemical synthesis, assembly, and cloning of a Mycoplasmagenitalium genome, Science, 2008, 319(5867): p.1215-20] and in vivo splicing ...
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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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