Gene Editing Service Technologies

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	High efficiency Large gene knockout/knockin capabilities Simultaneously targets multiple genes Applicable to many species Easy to construct Off-target effects can be reduced by choosing the appropriate sgRNA and eliminated by breeding; southern blot is used to screen out random insertions	Conventional KO mice/rats	5-7 months
		Conditional KO mice/rats
		ROSA26 locus gene KI mice/rats
		Gene KI/ Mutation mice/rats
		Humanized mice/rats
		Reporter/KO-then-cKO mice/rats
		Normal cell lines KO/KI
		hESC/iPS cells KO/KI
ESC/HR-based Gene Editing	Accurate, precise and reliably established The only targeting technology which can meet the requirements of almost all genome modifications Because ES cells are required, only mouse cells can be used for this technique Time consuming Heavy workload High cost	Conventional knockout (KO) mice	7-11 months
		Conditional KO mice
		ROSA26 locus gene knockin (KI) mice
		Gene KI/ Mutation mice
		Humanized mice
		Reporter/KO-then-cKO mice
Transgenic Technology	Fast timeline Low cost Super large fragment insertion Disadvantages include random insertion, instability, large number of founders that can cost more time and money	Transgenic mice/rats	2-3 months for F0
Chromosome Engineering	Gene cluster replacement at the megabase scale A unique technology for specific applications such as antibody gene cluster humanization	Mouse	Upon request
Other Services	In addition to full-scale gene editing services, Biocytogen also provides milestone services related to gene editing	CRISPR plasmid construction; CRISPR activity assay; Donor plasmid construction	Upon request