Gene Editing

Custom Genome Editing

Complete Pipeline

We deliver services tailored to your specific goals, including design and cloning of your gRNA and HR donor plasmids, as well as generation of stable single-clonal cell lines. We provide timely project updates and a comprehensive final report to keep you updated, and our scientists are always available to answer your questions.

Highlighted Features

Techniques

Permanent Knock-out
Conditional Knock-out
Point mutation
Insertion

Deliverables

2 vials of cells at 0.5~1X10⁶ cells/vial

Time Period

2-5 Months

Quality Control & Deliverables

Sanger; optional NGS; milestone updates

Background >>>> Click to view

Genome editing is a powerful tool to introduce additions, deletions, and substitutions in the genome of a living organism. The development of genome editing technologies involves with the use of meganucleases; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); and, most recently, the CRISPR/Cas9 system (Figure 1).

Meganucleases are nucleases with very long DNA-binding recognition sites which are from 14 to 40 nucleotides. Due to its highly specific and long recognition sequence, it is not applicable in modification on a target site in the genome.
ZFN is an artificial restriction enzyme that is composed of 6 to 8 DNA recognition motifs, or zinc fingers, and the catalytic domain of DNA-cleaving enzyme, FokI.
Similar to ZNFs, TALENs are composed of a DNA-cleaving protein fused with an array of nuclear effector domains.
CRISPR/Cas9 system is the most commonly used tool for genome editing currently. It has two components, a ~20 nucleotides small guide RNA (SgRNA) which binds to target DNA sequence and a Cas9 protein which exhibits nuclease function.

Figure 1. Overview of meganuclease, ZFN, TALEN, and CRISPR/Cas9 editing approaches.

Our genome editing work flow

Case Study

1. Point Mutation

Human ApoE has three isoforms, ApoE2, ApoE3 and ApoE4. ApoE3 and ApoE4 differ from each another only at one amino acid residue at position 112. To facilitate the studies of AD-related pathologies in neurons induced specifically by ApoE4, as well as for drug discovery, we developed ApoE3 cell line from human iPS ApoE4 cell line using CRISPR/Cas9.

Figure 2. APOE homozygous point mutation. (A) Schematic representation of point mutation strategy. (B) Sequence confirmation of point mutation from ApoE4 to ApoE3 in human iPS cells.

2. Gene Knockout

An apolipoprotein E (APOE) knockout (KO) cell line can be an important tool for functional study of APOE since APOE has been shown to play critical roles in lipid metabolism and neurodegenerative disease. Here in this example, we used CRISPR/Cas 9 system to generate ApoE KO cell line.

Figure 3. Generation of APOE KO cell line. (A) Schematic representation of gene knockout strategy. (B) Image of PCR amplification. (C & D) APOE KO confirmed by Sanger sequencing and western blotting results.

3. Gene Knockin

Neurogenin (Ngn) 2 is a basic helix-loop-helix (bHLH) transcription factor that promotes direct differentiation of pluripotent stem cells to functional excitatory neurons. Precise insertion of tetracycline inducible Ngn2 into human iPS cells at AAVS1 site could generate a stable iPSC line for rapid neuronal differentiation upon induction.

Figure 4. Knock in of Tet-on system into the AASV1 locus. (A) SgRNA was designed to target AASV1 locus. (B) Upon Dox treatment, human iPS cells were converted into excitatory neurons.

Figure 5. Upon doxycycline treatment, inducible Ngn2 iPS cells differentiated to neurons. (A) Absence of doxycycline. (B) 3 days after Dox. (C) 6 days after Dox. (D) 9 days after Dox - cells were beta-III-tubulin (Tuj) positive.

Publications

Bertholet, AM., et al. H⁺ transport is an integral function of the mitochondrial ADP/ATP carrier. Nature (2019). 571, p515–520
Diggins, NL., et al. α5β1 integrin trafficking and Rac activation are regulated by APPL1 in a Rab5-dependent manner to inhibit cell migration. Journal of Cell Science (2018) 131, jcs207019.
Shahid, M., et al. Centromere protein F (CENPF), a microtubule binding protein, modulates cancer metabolism by regulating pyruvate kinase M2 phosphorylation signaling. Cell Cycle (2018). 17, 24, p2802–2818
Wu, M., et al. Conditional gene knockout and reconstitution in human iPSCs with an inducible Cas9 system. Stem Cell Research (2018). 29, p6-14

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