Use of CRISPR/Cas ribonucleoproteins for high throughput gene editing of induced pluripotent stem cells

Wang, Qi; Chear, Sueanne; Wing, Kristof; Stellon, David; Tran, Minh Thuan Nguyen; Talbot, Jana; Pébay, Alice; Hewitt, Alex W.; Cook, Anthony

doi:10.1016/j.ymeth.2021.02.009

Cited by 11 publications

(4 citation statements)

References 44 publications

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“…In our CRISPR/Cas9 experiments, we found that the efficiency of single nucleotide editing varied between transfections. Our rate of HDR at the level of the bulk population was lower [5,7,16] or similar [39] to that of previously published optimisation reports in which bulk population data were analysed. The lower efficiency in our iPSC experiments can be explained by the suboptimal cut-tomutation distance which is determined by the location of the gRNA.…”

Section: Discussionsupporting

confidence: 89%

An efficient and adaptable workflow for editing disease-relevant single nucleotide variants using CRISPR/Cas9

Usher

Ligammari

Ahrabi

et al. 2021

Preprint

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Single nucleotide variants are the commonest genetic alterations in the human genome. At least 60,000 have been reported to be associated with disease. The CRISPR/Cas9 system has transformed genetic research, making it possible to edit single nucleotides and study the function of genetic variants in vitro. While significant advances have improved the efficiency of CRISPR/Cas9, the editing of single nucleotides remains challenging. There are two major obstacles: low efficiency of accurate editing and the isolation of these cells from a pool of cells with other editing outcomes. We present data from 85 transfections of induced pluripotent stem cells and an immortalised cell line, comparing the effects of altering CRISPR/Cas9 design and experimental conditions on rates of single nucleotide substitution. We targeted variants in TP53, which predispose to several cancers, and in TBXT which is implicated in the pathogenesis of the bone cancer, chordoma. We describe a scalable and adaptable workflow for single nucleotide editing that incorporates contemporary techniques including Illumina MiSeq™ sequencing, TaqMan™ qPCR and digital droplet PCR for screening transfected cells as well as quality control steps to mitigate against common pitfalls. This workflow can be applied to CRISPR/Cas9 and other genome editing systems to maximise experimental efficiency.Simple SummaryCRISPR/Cas9 has revolutionised genetic research. Cas9 generates a double strand break with high efficiency which is repaired by a cell’s pathways. If a genetic template is provided, the damage can be accurately repaired to introduce a desired genetic alteration. However, accurate repair occurs at a low efficiency and in a small proportion of edited cells, representing the main obstacles in harnessing CRISPR’s full potential. Using data from 85 CRISPR experiments for single nucleotide editing, targeting three locations in the human genome that are implicated in predisposition to cancer, we report the effect of different experimental conditions on editing efficiency. We describe current technologies that can be used to streamline the identification of accurately edited cells and synthesise these into an adaptable workflow that can be applied to CRISPR/Cas9 experiments to achieve single nucleotide editing in disease-relevant cell models.

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Section: Discussionsupporting

confidence: 89%

An efficient and adaptable workflow for editing disease-relevant single nucleotide variants using CRISPR/Cas9

Usher

Ligammari

Ahrabi

et al. 2021

Preprint

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“…Editing of CLN3 to delete exons 7 and 8 was done as previously described 32,33 . Briefly, 8 x 10 5 H9 hESCs in single-cell suspension were resuspended in 100 µL electroporation buffer from the Human Stem Cell Nucleofector Solution 2 kit (Lonza, VPH-5022).…”

Section: Methodsmentioning

confidence: 99%

Genetic and cellular basis of impaired phagocytosis and photoreceptor degeneration in CLN3 disease

Han,

Chear,

Talbot

et al. 2024

Preprint

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PurposeCLN3 Batten disease (also known as Juvenile Neuronal Ceroid Lipofuscinosis; JNCL) is a lysosomal storage disorder that typically initiates with retinal degeneration but is followed by seizure onset, motor decline and premature death. Patient-derived CLN3 disease iPSC-RPE cells show defective phagocytosis of photoreceptor outer segments (POSs). Because modifier genes are implicated in CLN3 disease, our goal here was to investigate a direct link betweenCLN3mutation and POS phagocytosis defect.MethodsIsogenic control andCLN3mutant stem cell lines were generated by CRISPR-Cas9mediated biallelic deletion of exons 7 and 8. A transgenicCLN3Δ7-8/Δ7-8(CLN3) Yucatan miniswine was also used to study the impact ofCLN3Δ7-8/Δ7-8mutation on POS phagocytosis. POS phagocytosis by cultured RPE cells was analyzed by Western blotting and immunohistochemistry. Electroretinogram, optical coherence tomography and histological analysis ofCLN3Δ7/8and wild-type miniswine eyes were carried out at 6-, 36-, or 48-month age.ResultsCLN3Δ7-8/Δ7-8RPE (CLN3RPE) displayed reduced POS binding and consequently decreased uptake of POS compared to isogenic control RPE cells. Furthermore, wild-type miniswine RPE cells phagocytosedCLN3Δ7-8/Δ7-8POS less efficiently than wild-type POS. Consistent with decreased POS phagocytosis, lipofuscin/autofluorescence was decreased inCLN3miniswine RPE at 36 months-of-age and was followed by almost complete loss of photoreceptors at 48 months of age.ConclusionsCLN3Δ7-8/Δ7-8mutation (that affects up to 85% patients) affects both RPE and POSs and leads to photoreceptor cell loss in CLN3 disease. Furthermore, both primary RPE dysfunction and mutant POS independently contribute to impaired POS phagocytosis in CLN3 disease.

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“…Editing of CLN3 iPSCs to correct 966 bp deletion was done essentially as described [71]. Single cell suspension (800,000 cells) of CLN3 iPSCs was electroporated with Cas9-sgRNA ribonucleoprotein (IDT) and 966 bp donor plasmid using Amaxa 4D Nucleofector (Lonza) with program CB 150. iPSCs were treated with puromycin 72 hours after CRISPR/Cas9 electroporation when cells have reached ∼80% confluency.…”

Section: Methodsmentioning

confidence: 99%

Lysosomal alterations and decreased electrophysiological activity in CLN3 disease (966 bp deletion, E295K) patient-derived cortical neurons

Chear

Perry

Wilson

et al. 2022

Preprint

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CLN3 disease is a lysosomal storage disorder associated with fatal neurodegeneration that is caused by mutations in CLN3. Most individuals with CLN3 disease carry at least one allele with a 966 bp deletion in CLN3 which results in the deletion of exons 7 and 8. There is a need for more physiologically relevant human cell-based CLN3 disease models to better understand the cellular changes during the disease process. Using CRISPR/Cas9, we corrected the 966 bp deletion mutation in human induced pluripotent stem cells (iPSCs) of a compound heterozygous patient (CLN3 Δ 966 bp and E295K). The isogenic deletion-corrected and unedited CLN3 patient iPSCs were used for disease modeling. iPSC-derived neurons carrying this particular CLN3 mutation (CLN3 neurons) had lower functional activity as recorded using microelectrode arrays for most of the culture period. Proteomics analysis showed downregulation of proteins related to axon guidance and endocytosis at day in vitro (DIV) 14 and 42 in CLN3 neurons. This was accompanied by an increase in lysosomal-related proteins in CLN3 neurons. Western blot analysis revealed hyperglycosylation of the lysosomal marker, Lysosome Associated Membrane Protein 1 (LAMP1) in CLN3 neurons at DIV 14, 28 and 42, which was not apparent in control neurons. Ultrastructural analysis of CLN3 neurons showed numerous membrane-bound vacuoles containing diverse types of storage material, ranging from curvilinear deposits, multilamellar structures to osmiophilic deposits. Our findings suggest alterations in lysosomal function and neurodevelopment involving axon guidance and synaptic transmission in CLN3-deficient neuronal derivatives, which could be potential targets for therapy.

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Use of CRISPR/Cas ribonucleoproteins for high throughput gene editing of induced pluripotent stem cells

Cited by 11 publications

References 44 publications

An efficient and adaptable workflow for editing disease-relevant single nucleotide variants using CRISPR/Cas9

An efficient and adaptable workflow for editing disease-relevant single nucleotide variants using CRISPR/Cas9

Genetic and cellular basis of impaired phagocytosis and photoreceptor degeneration in CLN3 disease

Lysosomal alterations and decreased electrophysiological activity in CLN3 disease (966 bp deletion, E295K) patient-derived cortical neurons

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