STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells

Blanch-Asensio, Albert; Grandela, Catarina; Brandão, Karina O.; Korte, Tessa de; Mei, Hailiang; Ariyürek, Yavuz; Yiangou, Loukia; Mol, Mervyn P.H.; Meer, Berend J. van; Kloet, Susan L.; Mummery, Christine L.; Davis, Richard P.

doi:10.1016/j.crmeth.2022.100300

Cited by 26 publications

(19 citation statements)

References 66 publications

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“…( H ) Maximum number of reported SNVs generated in hiPSCs using serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation (STRAIGHT-IN; 11 variants)(40), dual-integrase cassette exchange (DICE; 14 variants) (9), single-stranded oligodeoxyribonucleic (ssODN; 20 variants) (38), and CRaTER (113 variants). Note that the ssODN approach generated homozygous SNVs, whereas DICE and CRaTER (as used here) generates cells with heterozygous SNVs.…”

Section: Resultsmentioning

confidence: 99%

“…MYH7 encodes myosin heavy chain β (MHC-β), a sarcomeric thick filament protein that is not expressed in hiPSCs (15). We use CRISPR/Cas9 to knock-in transgenes containing a promoterless partial MYH7 cDNA (exon [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] fused with a C-terminal fluorescent protein gene (eGFP or mTagBFP2) in cis with an antibiotic resistance cassette (PGK-PuroR or EF1α-BSD) into the endogenous MYH7 locus. Following standard antibiotics selection, we transiently overexpress MYH7 in hiPSCs using CRISPRa of the endogenous MYH7 promoter and sort for eGFP + /mTagBFP2 + cells using fluorescence-activated cell sorting (FACS), enabling 9-fold enrichment of desired heterozygous, biallelic editing.…”

Section: Introductionmentioning

confidence: 99%

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CRaTER enrichment for on-target gene-editing enables generation of variant libraries in hiPSCs

Friedman

Fayer

Pendyala

et al. 2023

Preprint

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Standard transgenic cell line generation requires screening 100-1000s of colonies to isolate correctly edited cells. We describe CRISPRa On-Target Editing Retrieval (CRaTER) which enriches for cells with on-target knock-in of a cDNA-fluorescent reporter transgene by transient activation of the targeted locus followed by flow sorting to recover edited cells. We show CRaTER recovers rare cells with heterozygous, biallelic-editing of the transcriptionally-inactive MYH7 locus in human induced pluripotent stem cells (hiPSCs), enriching on average 25-fold compared to standard antibiotic selection. We leveraged CRaTER to enrich for heterozygous knock-in of a library of single nucleotide variants (SNVs) in MYH7, a gene in which missense mutations cause cardiomyopathies, and recovered hiPSCs with 113 different MYH7 SNVs. We differentiated these hiPSCs to cardiomyocytes and show MYH7 fusion proteins can localize as expected. Thus, CRaTER substantially reduces screening required for isolation of gene-edited cells, enabling generation of transgenic cell lines at unprecedented scale.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

CRaTER enrichment for on-target gene-editing enables generation of variant libraries in hiPSCs

Friedman

Fayer

Pendyala

et al. 2023

Preprint

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“…Finally, so as to apply this design to a wide range of encapsulated cell therapies, a stable insertion of DNA would be required instead of transient transfection. For such purposes, different systems have been described that allow the stable installation of multiple genes in different cell lines [46][47][48] . In addition, since the final product would involve genetically modified cells, it would have to fulfill the biosafety requirements that apply to genetically modified organisms used in the clinic 49 .…”

Section: Assembly and Functional Validation Of The Material-genetic I...mentioning

confidence: 99%

Engineering a material-genetic interface as safety switch for embedded therapeutic cells

Jerez‐Longres

Gómez-Matos

Becker

et al. 2023

Preprint

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Encapsulated cell-based therapies involve the use of genetically-modified cells embedded in a material in order to produce a therapeutic agent in a specific location in the patient's body. This approach has shown great potential in animal model systems for treating diseases such as type I diabetes or cancer, with selected approaches having been tested in clinical trials. Despite the promise shown by encapsulated cell therapy, though, there are safety concerns yet to be addressed, such as the escape of the engineered cells from the encapsulation material and the resulting production of therapeutic agents at uncontrolled sites of the body. For that reason, there is great interest in the implementation of safety switches that protect from those side effects. Here, we develop a material-genetic interface as safety switch for engineered mammalian cells embedded into hydrogels. Our switch allows the therapeutic cells to sense whether they are embedded in the hydrogel by means of a synthetic receptor and signaling cascade that link transgene expression to the presence of an intact embedding material. The system design is highly modular, allowing its flexible adaptation to other cell types and embedding materials. This autonomously acting switch constitutes an advantage over previously described safety switches, which rely on user-triggered signals to modulate activity or survival of the implanted cells. We envision that the concept developed here will advance the safety of cell therapies and facilitate their translation to clinical evaluation.

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“…Transfection of these iPSCs with serine integrase Bxb1 and DNA cargo containing attB sites allowed for integration of extremely large sequences such as a 173 kb bacterial artificial chromosome. 45 Through clever inclusion of unique loxP sites surrounding their landing pad, exogenous administration of Cre-recombinase after integration led to an almost "scarless" integration through removal of residual landing pad sequences. One could utilize this system to generate a barcoded library of specific variants and integrate into the recipient cells, induce differentiation toward a desired cell type and monitor the frequencies of variants in the population overtime.…”

Section: Targeted Integration Of Large Dna Cargomentioning

confidence: 99%

“…Yet another exciting targeted large payload integration method was recently described by Blanch‐Asensio et al 45 The method, called STRAIGHT‐IN, utilizes a nuclease‐free approach and achieves high efficiency and accuracy in gene targeting in human iPSCs (Figure 1C). The authors report successful insertion of DNA sequences up to 100 kb in size, which is significantly larger than what is achievable using traditional methods.…”

Section: Targeted Integration Of Large Dna Cargomentioning

confidence: 99%

Modeling human cancer predisposition syndromes using CRISPR/Cas9 in human cell line models

Draper

Panken

Largaespada

2023

Genes Chromosomes & Cancer

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The advancement of CRISPR mediated gene engineering provides an opportunity to improve upon preclinical human cell line models of cancer predisposing syndromes. This review focuses on using CRISPR/Cas9 genome editing tools to model various human cancer predisposition syndromes. We examine the genetic mutations associated with neurofibromatosis type 1, Li‐Fraumeni syndrome, Gorlin syndrome, BRCA mutant breast and ovarian cancers, and APC mutant cancers. Furthermore, we discuss the possibilities of using next‐generation CRISPR‐derived precision gene editing tools to introduce a variety of genetic lesions into human cell lines. The goal is to improve the quality of preclinical models surrounding these cancer predisposition syndromes through dissecting the effects of these mutations on the development of cancer and to provide new insights into the underlying mechanisms of these cancer predisposition syndromes. These studies demonstrate the continued utility and improvement of CRISPR/Cas9‐induced human cell line models in studying the genetic basis of cancer.

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STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells

Cited by 26 publications

References 66 publications

CRaTER enrichment for on-target gene-editing enables generation of variant libraries in hiPSCs

CRaTER enrichment for on-target gene-editing enables generation of variant libraries in hiPSCs

Engineering a material-genetic interface as safety switch for embedded therapeutic cells

Modeling human cancer predisposition syndromes using CRISPR/Cas9 in human cell line models

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