Applying a CRISPR/Cas9 recombineering strategy for multi-kilobase synthetic DNA sequence assembly in the E.coli chromosome and subsequent genomic replacement in mouse embryonic stem cells.文献综述

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