Site-specific gene modification usually uses homologous recombination of endogenous DNA with exogenous DNA. Homologous recombination is a natural genetic recombination event in which specific nucleotide sequences are randomly exchanged between similar or identical residues.
In advancing genetic engineering, we as scientists have learned to utilize the phenomena of homologous recombination to modify or gene-edit mouse embryonic stem cells (ESC). Gene targeting technology has evolved with the emergence of DNA nuclease-based gene-editing, including Zinc Finger Nuclease (ZFN), and more recent CRISPR/Cas9 technology, which allows even more precise and intentional genetic changes. Compared to ZFN and TALEN, CRISPR/Cas9 technology is more efficient, shortens the timeline/reduces the cost of production, breaks species constraints, and has the potential to directly edit genes in patient tissues.
Due to the typical results of experimentally low rates of homologous recombination, Biocytogen developed an innovative gene targeting technology: the CRISPR/Cas9-based Extreme Genome Editing — EGE™ — system, which can knock in large DNA fragments (> 5 kb) in the genomes of mice, rats and cell lines up-to 20-fold more efficiently than conventional CRISPR/Cas9 methods. EGE™ is successful for 98% of projects, which minimizes the timeline to generate genetically engineered animal and cell models.
|Technology||Advantages & Disadvantages||Applications||Timeline|
|CRISPR/EGE™-based Gene Editing||
||Conventional KO mice/rats||5-7 months|
|Conditional KO mice/rats|
|ROSA26 locus gene KI mice/rats|
|Gene KI/ Mutation mice/rats|
|Normal cell lines KO/KI|
|hESC/iPS cells KO/KI|
|ESC/HR-based Gene Editing||
||Conventional knockout (KO) mice||7-11 months|
|Conditional KO mice|
|ROSA26 locus gene knockin (KI) mice|
|Gene KI/ Mutation mice|
||Transgenic mice/rats||2-3 months for F0|
||CRISPR plasmid construction; CRISPR activity assay; Donor plasmid construction||Upon request|