Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.
AuthorsKim S, Kim D, Cho SW, Kim J, Kim JS
Journal
PubMed ID24696461
'RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants, but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide ... More
Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.
AuthorsLiang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, Carte J, Chen W, Roark N, Ranganathan S, Ravinder N, Chesnut JD,
Journal
PubMed ID26003884
'CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that can limit overall efficiency and ease of use. Here we describe methods for rapid synthesis of ... More
Precise Correction of Heterozygous SHOX2 Mutations in hiPSCs Derived from Patients with Atrial Fibrillation via Genome Editing and Sib Selection.
AuthorsSumer SA, Hoffmann S, Laue S, Campbell B, Raedecke K, Frajs V, Clauss S, Kääb S, Janssen JWG, Jauch A, Laugwitz KL, Dorn T, Moretti A, Rappold GA
JournalStem Cell Reports
PubMed ID32976766
'Patient-specific human induced pluripotent stem cells (hiPSCs) offer unprecedented opportunities for the investigation of multigenic disease, personalized medicine, and stem cell therapy. For heterogeneous diseases such as atrial fibrillation (AF), however, precise correction of the associated mutation is crucial. Here, we generated and corrected hiPSC lines from two AF patients ... More
Efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of CRISPR-Cas9.
'Myeloid cells play critical and diverse roles in mammalian physiology, including tissue development and repair, innate defense against pathogens, and generation of adaptive immunity. As cells that show prolonged recruitment to sites of injury or pathology, myeloid cells represent therapeutic targets for a broad range of diseases. However, few approaches ... More
Precise and error-prone CRISPR-directed gene editing activity in human CD34+ cells varies widely among patient samples.
AuthorsModarai SR, Kanda S, Bloh K, Opdenaker LM, Kmiec EB
JournalGene Ther
PubMed ID32873924
'Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated CRISPR-associated nucleases (Cas) are among the most promising technologies for the treatment of hemoglobinopathies including Sickle Cell Disease (SCD). We are only beginning to identify the molecular variables that influence the specificity and the efficiency of CRISPR- directed gene editing, ... More
Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA.
AuthorsLiang X, Potter J, Kumar S, Ravinder N, Chesnut JD
JournalJ Biotechnol
PubMed ID27845164
'While CRISPR-based gene knock out in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step to seamless genome editing. Under the conditions described here, we achieved up to 56% targeted integration efficiency with up ... More
Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.
AuthorsRoberts B, Hendershott MC, Arakaki J, Gerbin KA, Malik H, Nelson A, Gehring J, Hookway C, Ludmann SA, Yang R, Haupt A, Grancharova T, Valencia V, Fuqua MA, Tucker A, Rafelski SM, Gunawardane RN
JournalStem Cell Reports
PubMed ID30956114
'We describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for ... More
Ribonucleoprotein Transfection for CRISPR/Cas9-Mediated Gene Knockout in Primary T Cells.
AuthorsOh SA, Seki A, Rutz S
JournalCurr Protoc Immunol
PubMed ID30334617
'CRISPR/Cas9 has enabled the rapid and efficient generation of gene knockouts across various cell types of several species. T cells are central players in adaptive immune responses. Gene editing in primary T cells not only represents a valuable research tool, but is also critical for next generation immunotherapies, such as ... More
Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells.
AuthorsSeki A, Rutz S
JournalJ Exp Med
PubMed ID29436394
CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great ... More
Gene-edited stem cells enable CD33-directed immune therapy for myeloid malignancies.
AuthorsBorot F, Wang H, Ma Y, Jafarov T, Raza A, Ali AM, Mukherjee S
JournalProc Natl Acad Sci U S A
PubMed ID31138698
Antigen-directed immunotherapies for acute myeloid leukemia (AML), such as chimeric antigen receptor T cells (CAR-Ts) or antibody-drug conjugates (ADCs), are associated with severe toxicities due to the lack of unique targetable antigens that can distinguish leukemic cells from normal myeloid cells or myeloid progenitors. Here, we present an approach to ... More
NK cells switch from granzyme B to death receptor-mediated cytotoxicity during serial killing.
AuthorsPrager I, Liesche C, van Ooijen H, Urlaub D, Verron Q, Sandström N, Fasbender F, Claus M, Eils R, Beaudouin J, Önfelt B, Watzl C
JournalJ Exp Med
PubMed ID31270246
NK cells eliminate virus-infected and tumor cells by releasing cytotoxic granules containing granzyme B (GrzB) or by engaging death receptors that initiate caspase cascades. The orchestrated interplay between both cell death pathways remains poorly defined. Here we simultaneously measure the activities of GrzB and caspase-8 in tumor cells upon contact ... More
CRISPR-engineered T cells in patients with refractory cancer.
AuthorsStadtmauer EA, Fraietta JA, Davis MM, Cohen AD, Weber KL, Lancaster E, Mangan PA, Kulikovskaya I, Gupta M, Chen F, Tian L, Gonzalez VE, Xu J, Jung IY, Melenhorst JJ, Plesa G, Shea J, Matlawski T, Cervini A, Gaymon AL, Desjardins S, Lamontagne A, Salas-Mckee J, Fesnak A, Siegel DL, Levine BL, Jadlowsky JK, Young RM, Chew A, Hwang WT, Hexner EO, Carreno BM, Nobles CL, Bushman FD, Parker KR, Qi Y, Satpathy AT, Chang HY, Zhao Y, Lacey SF, June CH
JournalScience
PubMed ID32029687
CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding ... More
Engineering the mode of morphogenetic signal presentation to promote branching from salivary gland spheroids in 3D hydrogels.
AuthorsSamuel RZ, Lei P, Nam K, Baker OJ, Andreadis ST
JournalActa Biomater
PubMed ID31988042
Previously we developed a fibrin hydrogel (FH) decorated with laminin-111 peptides (L
Genomic profiling of T-cell activation suggests increased sensitivity of memory T cells to CD28 costimulation.
AuthorsGlinos DA, Soskic B, Williams C, Kennedy A, Jostins L, Sansom DM, Trynka G
JournalGenes Immun
PubMed ID33223527
T-cell activation is a critical driver of immune responses. The CD28 costimulation is an essential regulator of CD4 T-cell responses, however, its relative importance in naive and memory T cells is not fully understood. Using different model systems, we observe that human memory T cells are more sensitive to CD28 ... More