Gene editing in hPSCs
|Reporter Line Generation on hPSCs||Using CRIPSR/CAS9 and donor plasmid to knock in reporter cassettes of your gene of interest, such as knock in GFP/tdTomato into tissue specific genes locus.||Three to four months|
|Gene Knockout on hPSCs||Using CRIPSR/CAS9 to introduce frame shift mutations to create gene knockout hPSC lines.||Two months|
|SNP Knock in on hPSCs||Using CRIPSR/CAS9 and ssODN to introduce point mutation (or SNP) in WT iPSCs, or for disease-associated point mutations correction in patient iPSCs.||Two months|
|Inducible Gene Knockout on hPSCs||iCRISPR hPSC line generation or “Cre-loxP” knock in strategy.||Three to six months|
|Inducible Gene Overexpression on hPSCs||Knock in “tet-on system” and gene of interest into the AAVS1 safe-harbor in hPSCs.||Three to six months|
|Customer Knock in on hPSCs||Due to the complexity of the projects, we may need to make a customer knock in strategy.||Three to six months|
Reporter Line Generation on hPSCs
1. Design sgRNA targets and donor plasmid construction
Our donor plasmid is usually constructed using the Gibson cloning method, which contains the tag (such as GFP, mcherry, tdTomato etc), and loxped selection maker (such as loxp-PGK-puro-loxp). The donor plasmid works with the specific sgRNA target together to achieve the HDR in hPSCs.
2. Plasmid electroporation on hPSCs
An example showing GFP electroporation on hPSCs. The electroporation efficiency is ~50%-90%.
3. Drug selection on 6-well plates
Gene Knockout on hPSCs
In our hands, the efficiency of CRIPSR induced biallelic gene frameshift is generally high (>50%), although there is big variation (20%-95%) among different genes or different targets.
SNP Knock in on hPSCs
CRIPSR based HDR is low. The SNP induction cases we got were all homozygous SNP induction clones. Alternatively, we provide “base editor” for the cases that match distinct standards (The Director will decide and discuss for each case).