Combining Single Strand Oligodeoxynucleotides and CRISPR/Cas9 to Correct Gene Mutations in β-Thalassemia-induced Pluripotent Stem Cells

Niu, Xiaohua; He, Wenyin; Song, Bing; Ou, Zhanhui; Fan, Di; Chen, Yu‐Chang; Fan, Yong; Sun, Xiaofang

doi:10.1074/jbc.m116.719237

Cited by 82 publications

(64 citation statements)

References 33 publications

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“…The detected unintended edits reported here are on-target edits, however Xdrop™ may also be applied for enrichment of bioinformatically identified candidate distal off-targets such as tumour suppressor genes or for unbiased analysis of insert integration patterns whether mediated by CRISPR, virus or other genetic engineering technologies. (4) and positive droplets are (5) identified by their fluorescence and physically separated from negative droplets using a standard cell sorter. DNA is released from double emulsion droplets (6) resulting in a population of long single DNA molecules enriched for the ROI and comprising kilobases of information.…”

Section: Discussionmentioning

confidence: 99%

Verification of CRISPR editing and finding transgenic inserts by Xdrop™ Indirect sequence capture followed by short- and long- read sequencing

Blöndal¹,

Gamba²,

Møller³

et al. 2020

Preprint

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Validation of CRISPR-Cas9 editing typically explore the immediate vicinity of the target site and distal off-target sequences, which have led to the conclusion that CRISPR-Cas9 editing is very specific. However, an increasing number of studies suggest that on-target unintended editing events like deletions and insertions are relatively frequent but unfortunately often missed in the validation of CRISPR-Cas9 editing. The deletions may be several kb and only affect one allele. The golden standard in molecular validation of gene editing is direct sequencing of relatively short PCR amplicons. This approach will allow detection of smaller editing events but will fail to detect larger rearrangements in particular when these only affect one of the two alleles. Detection of larger rearrangements requires that an extended region is analyzed and the characterization of events may benefit from long-read sequencing. Here we present a new microfluidic technology Xdrop™ that allows targeted enrichment of longer regions (~ 100 kb) using just a standard PCR primer set. We sequenced the enriched region on long-and short-read sequencing platforms to unravel unknown and unintended genome edits resulting from CRISPR-Cas9 gene editing. We show how loss of heterozygosity, due to an accidental insertion in one of the alleles, remained undetected using standard procedures but was discovered implementing the Xdrop™ system. This demonstrates the potential of such technology in CRISPR gene editing assessments, providing an extremely accurate tool.

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Section: Discussionmentioning

confidence: 99%

Verification of CRISPR editing and finding transgenic inserts by Xdrop™ Indirect sequence capture followed by short- and long- read sequencing

Blöndal¹,

Gamba²,

Møller³

et al. 2020

Preprint

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“…HBB mutations in the patient-derived iPSCs were completely corrected, with increased production of HPCs. In another study, mutations of CD41/42 (-CTTT) in iPSCs from a -thalassemia patient were successfully corrected using a combination of single-strand oligodeoxynucleotides with CRISPR/Cas9 [45,49]. The corrected iPSCs were selected for erythroblast differentiation and manifested restored expression of HBB protein.…”

Section: Direct Conversion Of Somatic Cells Into Hematopoietic Cellsmentioning

confidence: 99%

“…Since development of the CRISPR-Cas9 gene editing system [44], this technology evaluated for its potential to treat various genetic diseases [45 ■ ]. A number of groups have successfully applied CRISPR-Cas9 technology to correct -thalassemia mutations in patient-derived iPSCs [46][47][48][49]. A disease-causing mutation in the -globin gene (HBB) was corrected using CRISPR-Cas9 in iPSCs derived from a -thablassemia patient with minimal off-target effects [48].…”

Section: Direct Conversion Of Somatic Cells Into Hematopoietic Cellsmentioning

confidence: 99%

Generating autologous hematopoietic cells from human-induced pluripotent stem cells through ectopic expression of transcription factors

Hwang

Broxmeyer

Lee

2017

Current Opinion in Hematology

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Purpose of review Hematopoietic cell transplantation (HCT) is a successful treatment modality for patients with malignant and nonmalignant disorders, usually when no other treatment option is available. The cells supporting long-term reconstitution after HCT are the hematopoietic stem cells (HSCs), which can be limited in numbers. Moreover, finding an appropriate human leukocyte antigen-matched donor can be problematic. If HSCs can be stably produced in large numbers from autologous or allogeneic cell sources, it would benefit HCT. Induced pluripotent stem cells (iPSCs) established from patients’ own somatic cells can be differentiated into hematopoietic cells in vitro. This review will highlight recent methods for regulating human (h) iPSC production of HSCs and more mature blood cells. Recent findings Advancements in transcription factor-mediated regulation of the developmental stages of in-vivo hematopoietic lineage commitment have begun to provide an understanding of the molecular mechanism of hematopoiesis. Such studies involve not only directed differentiation in which transcription factors, specifically expressed in hematopoietic lineage-specific cells, are overexpressed in iPSCs, but also direct conversion in which transcription factors are introduced into patient-derived somatic cells which are dedifferentiated to hematopoietic cells. As iPSCs derived from patients suffering from genetically mutated diseases would express the same mutated genetic information, CRISPR-Cas9 gene editing has been utilized to differentiate genetically corrected iPSCs into normal hematopoietic cells. Summary IPSCs provide a model for molecular understanding of disease, and also may function as a cell population for therapy. Efficient differentiation of patient-specific iPSCs into HSCs and progenitor cells is a potential means to overcome limitations of such cells for HCT, as well as for providing in-vitro drug screening templates as tissue-on-a-chip models.

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“…This method is routinely performed in early embryos of many animal species [74][75][76]. Although this method was reported to have been applied to mutation correction in some patientderived iPSCs [50,77,78], it is not efficient in human cultured cells because of the less efficient HDR activity. Therefore, it is necessary to design experimental procedures for the concentration of ssODN-knock-in cells.…”

Section: Ssodn-mediated Single Nucleotide Variation (Snv) Knock-inmentioning

confidence: 99%

Updated summary of genome editing technology in human cultured cells linked to human genetics studies

2017

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Current deep-sequencing technology provides a mass of nucleotide variations associated with human genetic disorders to accelerate the identification of causative mutations. To understand the etiology of genetic disorders, reverse genetics in human cultured cells is a useful approach for modeling a disease in vitro. However, gene targeting in human cultured cells is difficult because of their low activity of homologous recombination. Engineered endonucleases enable enhancement of the local activation of DNA repair pathways at the human genome target site to rewrite the desired sequence, thereby efficiently generating disease-modeling cultured cell clones. These edited cells can be used to explore the molecular functions of a causative gene product to uncover the etiological mechanisms. The correction of mutations in patient cells using genome editing technology could contribute to the development of unique gene therapies. This technology can also be applied to screening causative mutations. Rare genetic disorders and non-exonic mutation-caused diseases remain frontier in the field of human genetics as it is difficult to validate whether the extracted nucleotide variants are mutation or polymorphism. When isogenic human cultured cells with a candidate variant reproduce the pathogenic phenotypes, it is confirmed that the variant is a causative mutation.

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Combining Single Strand Oligodeoxynucleotides and CRISPR/Cas9 to Correct Gene Mutations in β-Thalassemia-induced Pluripotent Stem Cells

Cited by 82 publications

References 33 publications

Verification of CRISPR editing and finding transgenic inserts by Xdrop™ Indirect sequence capture followed by short- and long- read sequencing

Verification of CRISPR editing and finding transgenic inserts by Xdrop™ Indirect sequence capture followed by short- and long- read sequencing

Generating autologous hematopoietic cells from human-induced pluripotent stem cells through ectopic expression of transcription factors

Updated summary of genome editing technology in human cultured cells linked to human genetics studies

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