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1. CGMP Compliant Microfluidic Transfection of Induced Pluripotent Stem Cells for CRISPR-Mediated Genome Editing

   https://doi.org/10.1093/stmcls/sxad063  

   Bohrer, Laura_R; Stone, Nicholas_E; Wright, Allison_T; Han, Sewoon; Sicher, Ian; Sulchek, Todd_A; Mullins, Robert_F; Tucker, Budd_A (August 2023, Stem Cells)

   Abstract Inherited retinal degeneration is a term used to describe heritable disorders that result from the death of light sensing photoreceptor cells. Although we and others believe that it will be possible to use gene therapy to halt disease progression early in its course, photoreceptor cell replacement will likely be required for patients who have already lost their sight. While advances in autologous photoreceptor cell manufacturing have been encouraging, development of technologies capable of efficiently delivering genome editing reagents to stem cells using current good manufacturing practices (cGMP) are needed. Gene editing reagents were delivered to induced pluripotent stem cells (iPSCs) using a Zephyr microfluidic transfection platform (CellFE). CRISPR-mediated cutting was quantified using an endonuclease assay. CRISPR correction was confirmed via digital PCR and Sanger sequencing. The resulting corrected cells were also karyotyped and differentiated into retinal organoids. We describe use of a novel microfluidic transfection platform to correct, via CRISPR-mediated homology-dependent repair (HDR), a disease-causing NR2E3 mutation in patient-derived iPSCs using cGMP compatible reagents and approaches. We show that the resulting cell lines have a corrected genotype, exhibit no off-target cutting, retain pluripotency and a normal karyotype and can be differentiated into retinal tissue suitable for transplantation. The ability to codeliver CRISPR/Cas9 and HDR templates to patient-derived iPSCs without using proprietary transfection reagents will streamline manufacturing protocols, increase the safety of resulting cell therapies, and greatly reduce the regulatory burden of clinical trials. 

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2. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells

   https://doi.org/10.1016/j.stemcr.2015.11.014  

   Carlson-Stevermer, Jared; Goedland, Madelyn; Steyer, Benjamin; Movaghar, Arezoo; Lou, Meng; Kohlenberg, Lucille; Prestil, Ryan; Saha, Krishanu (January 2016, Stem Cell Reports)
3. CRISPR–Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells

   https://doi.org/10.1093/nar/gkac049  

   Lin-Shiao, Enrique; Pfeifer, Wolfgang G.; Shy, Brian R.; Saffari Doost, Mohammad; Chen, Evelyn; Vykunta, Vivasvan S.; Hamilton, Jennifer R.; Stahl, Elizabeth C.; Lopez, Diana M.; Sandoval Espinoza, Cindy R.; et al (February 2022, Nucleic Acids Research)

   Abstract DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR–Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei. 

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4. Robust genome editing via modRNA-based Cas9 or base editor in human pluripotent stem cells

   https://doi.org/10.1016/j.crmeth.2022.100290  

   Haideri, Tahir; Howells, Alessandro; Jiang, Yuqian; Yang, Jian; Bao, Xiaoping; Lian, Xiaojun Lance (September 2022, Cell Reports Methods)
5. Application of CRISPR/Cas9-Mediated Gene Editing in Tomato

   Reem, N.T.; Van Eck, J. (June 2019, Methods in molecular biology)