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). With the emergence of DNA nuclease-based gene-editing, including Zinc Finger Nuclease (ZFN), and more recent CRISPR/Cas9 technology, even more precise and intentional genetic changes can be introduced. Compared with ZFN and TALEN, CRISPR/Cas9 technology is more efficient, shortens the timeline and reduces the cost of production, breaks species constraints, and also 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 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 for 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.
|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 to screen out random insertions.||Conventional KO mice/rats||6-8 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||Accurate, precise and reliably established, by far the only targeting technology which can meet the requirements of almost all genome modifications; but ES cells are required (only mouse cells are efficient for this technique), so it cannot be applied to other animal models); 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|
|Transgenic Technology||Traditional transgenic technology is welcomed because the fast timeline low cost, super large fragment insertion, etc. But as the advance of new gene targeting technologies, especially EGE™, above advantages are not very obvious anymore. Especially random insertion, instability, large number founders can cost more time and money sometimes||Transgenic mice/rats||2-3 months for F0|
|Chromosome Engineering||Gene cluster replacement at the scale of megabase. A unique technology for specific applications such as antibody gene cluster humanization.||Mouse||Upon request|
|Others Services||Besides the full-scale gene editing services, Biocytogen also provide milestone services relating with gene editing.||CRISPR plasmid construction; CRISPR activity assay; Donor plasmid construction||Upon request|