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Integration of xeno-free single-cell cloning in CRISPR-mediated DNA editing of human iPSCs improves homogeneity and methodological efficiency of cellular disease modeling - PubMed

  • ️Sun Jan 01 2023

Integration of xeno-free single-cell cloning in CRISPR-mediated DNA editing of human iPSCs improves homogeneity and methodological efficiency of cellular disease modeling

Atefeh Namipashaki et al. Stem Cell Reports. 2023.

Abstract

The capability to generate induced pluripotent stem cell (iPSC) lines, in tandem with CRISPR-Cas9 DNA editing, offers great promise to understand the underlying genetic mechanisms of human disease. The low efficiency of available methods for homogeneous expansion of singularized CRISPR-transfected iPSCs necessitates the coculture of transfected cells in mixed populations and/or on feeder layers. Consequently, edited cells must be purified using labor-intensive screening and selection, culminating in inefficient editing. Here, we provide a xeno-free method for single-cell cloning of CRISPRed iPSCs achieving a clonal survival of up to 70% within 7-10 days. This is accomplished through improved viability of the transfected cells, paralleled with provision of an enriched environment for the robust establishment and proliferation of singularized iPSC clones. Enhanced cell survival was accompanied by a high transfection efficiency exceeding 97%, and editing efficiencies of 50%-65% for NHEJ and 10% for HDR, indicative of the method's utility in stem cell disease modeling.

Keywords: CRISPR-Cas9 editing; clonal homogeneity; electroporatio; human iPSC; lipofection; single-cell cloning; transfection.

Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1
Figure 1

RNP lipid delivery achieves high cell survival and transfection rate (A) Flow cytometry analysis of CRISPRed iPSCs transfected using the lipid-based CRISPR delivery system shows a substantially higher degree of cell viability relative to electroporated cells, exceeding 40% (p = 0.0001). (B) Flow cytometry dot plot demonstrates that lipid RNP transfection using Lipofectamine Stem Transfection Reagent achieves close to 100% transfection efficiency (yellow and green) across three independent iPSC lines. The ATTO 550 (x axis) was plotted against a short-wavelength laser channel (488; y axis) to achieve a clear separation of the negative and positive population. Un-transfected cells are shown in purple (<3%). Reproducibility of the transfection efficiencies were tested across three independent experiments.

Figure 2
Figure 2

Integration of RNP lipid delivery with our unique method of single-cell cloning achieves highly improved homogeneous CRISPRed-iPSC clone survival (A) Gradual optimization method shows utilization of a rich environment containing StemFlex and CloneR supplement in wells coated with Laminin-521 achieves the highest single-cell clonal emergence following CRISPR transfection of up to 70.1%. (B) Application of the StemFlex+CloneR+Laminin-521 plating conditions in three different cell lines led to consistent high-level survival in CRISPRed (61.8%–70.1%) and non-CRISPRed single-cell clones (86.4%–90.2%). (C) Microscopy images showing a single-cell clone growth over time. Cells were imaged via the fluorescent NucBlue Live cell stain.

Figure 3
Figure 3

Our method demonstrates successful genome editing (A) Electropherograms of T7EN1 digestion on the pool of FACS-sorted positively transfected cells reveals an editing frequency in the range of 48.6%–57.5% in the targeted loci (which corresponds to cell pool columns in part B of the figure). The green (full length) and red (cleaved fragments) peaks are color coded for easy identification. RFU, relative fluorescence unit; LM, lower marker; UM, upper marker. (B) Editing frequency measured from 50 post-transfected single-cell clones (using Sanger sequencing) shows a range of 50.0%–65.0%, which is consistent to their respective cell pool measured by T7EN1 assay. Results are shown as mean of three independent experiments ±SEM.

Figure 4
Figure 4

Application of our method in HDR shows the desired editing achieved in 2 weeks The MICCNi002-A cell (AA genotype) were transfected with the RNP CRISPR-Cas9 complex targeting rs704074 (A/G) mapped to the DUSP6 gene using the donor molecule containing the G allele. The positively transfected single-cell clones were subjected to HRMA screening to identify the knocked in clones. Melt curves are normalized using the ratio between fluorescence and temperature variability (–d/dT). Of 50 screened clones, 5 were identified with different melting temperature (Tm) representing a successful HDR of 10%. This was also confirmed by DNA Sanger sequencing.

Figure 5
Figure 5

Schematic workflow for our genetically homogeneous CRISPRed-iPSC single-cell cloning Step 1: iPSCs are grown to 60%–80% confluency. Step 2: the ribonucleoprotein complex (RNP) consisting of gRNA:tracrRNA duplex and Cas9 protein is formed and transfected within the small clusters of iPSCs using Lipofectamine Stem Transfection Reagent (step 3). Step 4: following 48 h of incubation, positively transfected cells are single-cell sorted in a rich environment containing StemFlex medium and CloneR supplement in wells of 96-well plates coated with Laminin-521. Step 5: single cells are grown for 7–10 days until they form a homogeneous single-cell clone and reach sufficient confluency for expansion. Step 6: a portion of the clones is selected for detection and confirmation of the desired mutations (e.g., Sanger sequencing, western blotting) and the remainder utilized for follow-up phenotyping experiments (step 7). Figure created with

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