Biology subjects

Jang, H.-K.

Publications and source records attributed to Jang, H.-K..

5 recordsLinked to original sources

bioRxiv2024

Detailed mechanisms for unintended large DNA deletions with CRISPR, base editors, and prime editors

CRISPR-Cas9 nucleases are versatile tools for genetic engineering cells and function by producing targeted double-strand breaks (DSBs) in the DNA sequence. However, the unintended production of large deletions (>100 bp) represents a challenge to the effective application of this genome-editing system. We optimized a long-range amplicon sequencing system and developed a k-mer sequence-alignment algorithm to simultaneously detect small DNA alteration events and large DNA deletions. With this workflow, we determined that CRISPR-Cas9 induced large deletions at varying frequencies in cancer cell lines, stem cells, and primary T cells. With CRISPR interference screening, we determined that end resection and the subsequent TMEJ [DNA polymerase theta-mediated end joining] repair process produce most large deletions. Furthermore, base editors and prime editors also generated large deletions despite employing mutated Cas9 "nickases" that produce single-strand breaks. Our findings reveal an important limitation of current genome-editing tools and identify strategies for mitigating unwanted large deletion events.

genetics↗

bioRxiv2022

MSH2 and MSH6 as size dependent cellular determinants for prime editing in human embryonic stem cells

Potential applications of precise genome editing in human pluripotent stem cells (hPSCs), not only for isogenic disease modeling but also for ex vivo stem cell therapy, have urged the application of diverse genome editing tools in hPSCs. However, unlike differentiated somatic cells, the unique cellular properties of hPSCs (e.g., high susceptibility to DNA damage and active DNA repair) largely determine the overall efficiency of editing tools. Considering high demand of prime editors (PE), mostly due to its broad editing coverage compared to base editors, it is important to characterize the key molecular determinants of PE efficiency in hPSCs. Herein, we showed that MSH2 and MSH6, two main components of the MutS complex of mismatch repair (MMR), are highly expressed in hPSCs and determine PE efficiency in an editing size-dependent manner. Importantly, loss of MSH2, which disrupts both MutS and MutS{beta} complexes, was found to dramatically improve the efficiency of PE from one base to 10 bases, up to 50 folds. In contrast, genetic perturbation of MSH6, which solely abrogates MutS activity, marginally improved the editing efficiency up to 3 base pairs. The size dependent effect of MSH2 or MSH6 on prime editing in hPSCs not only implies MMR is a major determinant of PE efficiency in hPSCs but also highlights the distinct roles of MutS and MutS{beta} in the outcome of genome editing.

molecular biology↗

bioRxiv2021

Therapeutic adenine base editing corrects nonsense mutation and improves visual function in a mouse model of Leber congenital amaurosis

Leber congenital amaurosis (LCA) is an inherited retinal degeneration that causes severe visual dysfunction in children and adolescents. In patients with LCA, pathogenic variants are evident in specific genes, such as RPE65, which are related to the functions of retinal pigment epithelium and photoreceptors. Base editing confers a way to correct pathogenic substitutions without double-stranded breaks in contrast to the original Cas9. In this study, we prepared dual adeno-associated virus vectors containing the split adenine base editors with trans-splicing intein (AAV-ABE) for in vivo adenine base editing in retinal degeneration 12 (rd12) mice, an animal model of LCA, which possess a nonsense mutation of C to T transition in the Rpe65 gene (p.R44X). AAV-ABE induced an A to G transition in retinal pigment epithelial cells of rd12 mice when injected into the subretinal space. The on-target editing was sufficient to recover wild-type mRNA, RPE65 protein, and light-induced electrical responses of retinal tissues. We suggest adenine base editing to correct pathogenic variants in the treatment of LCA.

genetics↗

bioRxiv2020

Efficient GNE myopathy disease modeling with mutation specific phenotypes in human pluripotent stem cells by base editors

Despite the great potential of disease modeling with the isogenic pairs of human pluripotent stem cells (hPSCs), the extremely low efficiency of precise gene editing in hPSCs remains a technical hurdle for this approach. Herein, we took advantage of currently available base editors (BEs) to epitomize the isogenic disease model from hPSCs. Using this method, we established 14 hPSCs that harbor point mutations on the GNE gene, including four different mutations found in GNE myopathy patients. Due to lesser activation of p53 by BEs than Cas9, a higher editing efficiency with BEs was achieved. Four different mutations in the epimerase or kinase domains of GNE revealed mutation-specific hyposialylation, which was closely correlated to pathological clinical phenotypes. These mutation-specific hyposialylation patterns were evident in GNE protein structure modeling. Furthermore, treatment with a drug candidate currently under clinical trials showed a mutation-specific drug response in GNE myopathy disease models. These data suggest that isogenic disease models from hPSCs using BEs could serve as a useful tool for mimicking the pathophysiology of GNE myopathy and for predicting drug responses.

developmental biology↗

bioRxiv2019

Scarless Enriched selection of Genome edited Human Pluripotent Stem Cells Using Induced Drug Resistance

An efficient gene editing technique for use in human pluripotent stem cells (hPSCs) would have great potential value in regenerative medicine, as well as in drug discovery based on isogenic human disease models. However, the extremely low efficiency of gene editing in hPSCs is a major technical hurdle that remains to be resolved. Previously, we demonstrated that YM155, a survivin inhibitor developed as an anti-cancer drug, induces highly selective cell death in undifferentiated hPSCs. In this study, we demonstrated that the high cytotoxicity of YM155 in hPSCs, which is mediated by selective cellular uptake of the drug, is due to high expression of SLC35F2 in these cells. Consistent with this, knockout of SLC35F2 with CRISPR-Cas9 or depletion with siRNAs made hPSCs highly resistant to YM155. Simultaneous gene editing of a gene of interest and transient knockdown of SLC35F2 following YM155 treatment enabled genome-edited hPSCs to survive because YM155 resistance was temporarily induced, thereby achieving enriched selection of genome-edited clonal populations. This precise and efficient genome editing approach took as little as 3 weeks without cell sorting or introduction of additional genes.

developmental biology↗