TY  - JOUR
AB  - STAT3-hyper IgE syndrome (STAT3-HIES) is a primary immunodeficiency presenting with destructive lung disease along with other symptoms. CRISPR-Cas9-mediated adenine base editors (ABEs) have the potential to correct one of the most common STAT3-HIES causing heterozygous STAT3 mutations (c.1144C>T/p.R382W). As a proof-of-concept, we successfully applied ABEs to correct STAT3 p.R382W in patient fibroblasts and induced pluripotent stem cells (iPSCs). Treated primary STAT3-HIES patient fibroblasts showed a correction efficiency of 29% ± 7% without detectable off-target effects evaluated through whole-genome and high-throughput sequencing. Compared with untreated patient fibroblasts, corrected single-cell clones showed functional rescue of STAT3 signaling with significantly increased STAT3 DNA-binding activity and target gene expression of CCL2 and SOCS3. Patient-derived iPSCs were corrected with an efficiency of 30% ± 6% and differentiated to alveolar organoids showing preserved plasticity in treated cells. In conclusion, our results are supportive for ABE-based gene correction as a potential causative treatment of STAT3-HIES.
AU  - Eberherr, A.C.
AU  - Maaske, A.
AU  - Wolf, C.
AU  - Giesert, F.
AU  - Berutti, R.
AU  - Rusha, E.
AU  - Pertek, A.
AU  - Kastlmeier, M.T.
AU  - Voss, C.
AU  - Plummer, M.
AU  - Sayed, A.
AU  - Graf, E.
AU  - Effner, R.
AU  - Volz, T.*
AU  - Drukker, M.
AU  - Strom, T.M.
AU  - Meitinger, T.
AU  - Stöger, T.
AU  - Buyx, A.M.*
AU  - Hagl, B.
AU  - Renner, E.D.
C1  - 61828
C2  - 50479
CY  - 140 Huguenot Street, 3rd Fl, New Rochelle, Ny 10801 Usa
SP  - 178-190
TI  - Rescue of STAT3 function in hyper-IgE syndrome using adenine base editing.
JO  - CRISPR J.
VL  - 4
IS  - 2
PB  - Mary Ann Liebert, Inc
PY  - 2021
SN  - 2573-1599
ER  - 

TY  - JOUR
AB  - Gene manipulations of human induced pluripotent stem cells (iPSCs) by CRISPR-Cas9 genome engineering are widely used for disease modeling and regenerative medicine applications. There are two competing pathways, non-homologous end joining (NHEJ) and homology directed repair (HDR) that correct the double-strand break generated by CRISPR-Cas9. Here, we improved gene editing efficiency of gene knock-in (KI) in iPSCs with minimum components by manipulating the Cas9 expression vector. Either we inserted short hairpin RNA expression cassettes to downregulate DNAPK and XRCC4, two main players of the NHEJ pathway, or we increased cell survival by inserting an anti-apoptotic expression cassette of miRNA-21 into the Cas9 vector. For an easy readout, the pluripotency gene SOX2 was targeted with a T2A-tdTomato reporter construct. In vitro downregulating DNAPK and XRCC4 increased the targeting efficiency of SOX2 KI by around twofold. Furthermore, co-expression of miRNA-21 and Cas9 improved the efficiency of SOX2 KI by around threefold. Altogether, our strategies provide a simple and valuable approach for efficient CRISPR-Cas9 gene editing in iPSCs.
AU  - Shahryari, A.
AU  - Moya, N.
AU  - Siehler, J.
AU  - Wang, X.
AU  - Burtscher, I.
AU  - Lickert, H.
C1  - 62853
C2  - 51105
CY  - 140 Huguenot Street, 3rd Fl, New Rochelle, Ny 10801 Usa
SP  - 491-501
TI  - Increasing gene editing efficiency for CRISPR-Cas9 by small RNAs in pluripotent stem cells.
JO  - CRISPR J.
VL  - 4
IS  - 4
PB  - Mary Ann Liebert, Inc
PY  - 2021
SN  - 2573-1599
ER  -