Abstract
For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector concurrently with episomal reprogramming plasmids into fibroblasts. Our experiments have produced nearly even numbers of all 3 genotypes in autosomal genes. In addition, we provide a detailed approach for maintaining and genotyping 96-well plates of iPSC clones.
Keywords: Induced pluripotent stem cells, Genome editing, Disease modeling, Cellular reprogramming, CRISPR/Cas9, Human fibroblasts
Background
CRISPR/Cas9 technology has allowed easy and specific targeting of a particular genomic location for gene-editing. Combining this technology with the disease modeling and regenerative medicine potential of induced pluripotent stem cells (iPSCs) will continue to have unprecedented impacts on biomedical research. However, adapting the CRISPR/Cas9 system to iPSCs has presented several challenges. The traditional approach for gene-editing in cell lines is to transfect the cells with a plasmid expressing the Cas9 protein and guide RNA (gRNA) after which single clones are generated and screened for the desired genetic alteration. Unfortunately, iPSCs are not amenable to single cell cloning. Several media supplements and cloning approaches have been developed to overcome this difficulty but are still fraught with either expensive equipment (low oxygen incubators), difficult technological steps (survival of FACS sorted single iPSCs), or labor intensive protocols (sub-cloning) (Forsyth et al., 2006; Miyaoka et al., 2014). Moreover, single cell passaging has been linked to increased genomic abnormalities in iPSCs (Bai et al., 2015). Fluorescent or antibiotic resistance genetic markers have been used to overcome issues with clonality and the overall low efficiency of gene-editing in these cells, but require homologous recombination of a large cassette via a targeting plasmid designed with long homology arms (400-800 bp) (Hendel et al., 2014). Designing these plasmids takes a great deal of time.To overcome many of these obstacles, we utilized a combined approach of simultaneous reprogramming and CRISPR/Cas9 mutagenesis to generate both heterozygous and homozygous loss-of-function (LOF) iPSC lines. This combined approach was first presented by Howden and colleagues for homologous recombination gene-editing (Howden et al., 2015 and 2016), but we have expanded upon and further defined their original results for indel formation in a recent publication (Tidball et al., 2017). This procedure takes advantage of the cloning step inherent to iPSC reprogramming as well as the greater ease of transfection in fibroblast culture (Figure 1). The ability to efficiently generate a large number of clones with a similar proportion of all three genotypes (wild-type/wild-type, wild-type/indel, and indel/indel) will allow rapid development of LOF iPSC lines for disease modeling and basic research. Figure 1. Overview of the experimental workflow. The protocols in this article are broken into 4 major sections, including: guide RNA and PCR primer design (Step 1), CRISPR plasmid generation and purification (Steps 2-6), fibroblast culture and electroporation with CRISPR and reprogramming plasmids (Steps 7-8), and isolating genomic DNA followed by PCR of the targeted region and sequencing (Steps 11-15).
Materials and Reagents
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Acknowledgments
This work was supported by grants from the NIHNS090364 and Citizens United for Research in Epilepsy (CURE) to J.M.P. We thank the Center for Computational and Integrative Biology (CCIB) DNA Core Facility at Massachusetts General Hospital (Cambridge, MA) for providing sequencing services. The authors do not have any financial or non-financial interest in the subject matter or materials discussed in this manuscript.
References
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