NHLBI iPSC Core Facility

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

In FY21, we saw significant increase of requests for CRISPR/Cas gene-editing services. Most of new requests were mutation knockin projects which are similar to gene-correction projects because they are both HDR-mediated gene-editing projects, but they also reflect that more NIH labs who dont have access to patient samples to generate patient-specific iPSCs now are interested in establishing iPSC models using mutation knockin approach. We also saw for the first time that several mutation knockin projects are heterozygous knockin projects, which are technically more challenging and designed for more sophisticated disease models. To meet the demand, we improve our gene-editing methods and efficiency so that in FY21 we were able to complete 30 projects, which are 50% more than what we did in FY20. Continuing using RNP transfection method and small molecules that enhance HDR efficiency and iPSC clone survival, we found we dont need to use FACS sorting anymore and can further improve heterozygous mutation knockin efficiency by adding dCas9. Besides achieving biallelic gene knockout at 20-90% efficiency, we can now accomplish precision gene correction or mutation knockin at 10-90% efficiency among which homozygous knockin tends to have higher efficiency than heterozygous knockin. We optimized our protocol so that we can generate gene knockout, homozygous and heterozygous knockin clones all in one experiment within 2-3 months, significantly saving time and money for our users. In FY21, we fully established gene-specific reporter knockin service with additional ddPCR genotyping confirmation and improved its efficiency from 1-10% to 10-90%, comparable to that in mutation knockin projects. In FY21 we finished 11 gene knockout projects, 10 gene correction projects, 8 mutation knockin projects, and 1 gene knockin in project. These iPSC gene-editing projects were done by Core Director and one full-time biologist, and 15 of them were for 3 NHLBI labs and 15 were for 3 labs from NEI, NHGRI, and NINDS. The genetically modified iPSC lines are being used as isogenic control lines to model many human diseases. In FY21, we continued to provide high-quality, integration-free iPSC generation services. Due to COVID-19 impact on our users research and budget, we only received and reprogrammed 42 blood samples and 45 skin fibroblast samples. Among 87 reprogramming projects in FY21, 20 were for 5 NHLBI labs and 67 were for 11 labs from NEI, NIMH, and NHGRI. The iPSC reprogramming projects were mostly done by one half-time biologist with the help from other Core staff. Since the Core was established in 2011, we have generated >940 iPSC lines. Working with Dunbar lab and Boehm lab in NHLBI, we generated 500 million iPSC-derived cardiomyocytes that have been used in autologous transplantation in the second animal for myocardial infraction nonhuman primate model. The animal survived well after transplantation and we have been monitoring the cells and heart function for more than 4 months. We are now preparing iPSC-cardiomyocytes for the third animal. In FY21, 10 NHLBI labs from 8 Branches/Centers in NHLBI received iPSC Core services, while 19 non-NHLBI labs from 9 ICs received our services. While only 16.5% cost recovery came from NHLBI labs, 54.1% of all the service projects were provided to NHLBI PIs. In FY21, we published 8 papers with NHLBI and NCATS labs (https://www.ncbi.nlm.nih.gov/myncbi/jizhong.zou.1/bibliography/public/ ). To contribute to broad research community, we have deposited our iPSC gene editing vectors in non-profit repository Addgene (https://www.addgene.org/Jizhong_Zou/ ), who has distributed our top 10 vectors for 645 times to 396 laboratories in 282 organizations worldwide.