开题报告内容Content：（包括拟研究或解决的问题、采用的研究手段及文献综述，不少于2000字）Including objectives, methods adopted and literature review, no less than 2000 words
Objectives:
Applying the CRISPR/Cas9 coupled recombineering strategy to assemble tracts of multi-kilobase human synthetic sequences in the E. coli chromosome, subsequent assembled DNA recovery from the chromosome and genomic replacement in mouse ES cells.
Methods:
DNA assembly in vitro: Oligodeoxynucleotide synthesis
Overlap extension PCR
Golden Gate assembly
Gibson assembly
E. coli genome engineering: Electroporation
Coupled CRISPR/Cas9 recombineering
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