Simultaneous reprogramming and gene editing of human fibroblasts

Abstract

The utility of human induced pluripotent stem cells (iPSCs) is enhanced by an ability to precisely modify a chosen locus with minimal impact on the remaining genome. However, the derivation of gene-edited iPSCs typically involves multiple steps requiring lengthy culture periods and several clonal events. Here, we describe a one-step protocol for reliable generation of clonally derived gene-edited iPSC lines from human fibroblasts in the absence of drug selection or FACS enrichment. Using enhanced episomal-based reprogramming and CRISPR/Cas9 systems, gene-edited and passage-matched unmodified iPSC lines are obtained following a single electroporation of human fibroblasts. To minimize unwanted mutations within the target locus, we use a Cas9 variant that is associated with decreased nonhomologous end-joining (NHEJ) activity. This protocol outlines in detail how this streamlined approach can be used for both monoallelic and biallelic introduction of specific base changes or transgene cassettes in a manner that is efficient, rapid (∼6-8 weeks), and cost-effective.

Figure 1

HDRCas9-Gem facilitates generation of indel-free gene-edited iPSCs. (a) Schematic diagram of ‘on-target’ indel mutations that are commonly observed in successfully targeted clones using CRISPR/Cas9. (b) Schematic diagram of the Cas9-Gem variant. An upstream T7 promoter is used to facilitate in vitro transcription. (c) Western blot analysis of Cas9-Gem and wtCas9 protein expression in sorted G0/G1 and S/G2 subpopulations shows enrichment of Cas9-Gem in the S/G2 subpopulation and lower levels in G1. Loading control is anti-actin. (d, Left) Proportion of heterozygous gene-targeted iPSC clones identified without disruption of the second allele following knock-in of an EGFP reporter within the DNMT3B locus using either wtCas9 (n = 17) or Cas9-Gem (n = 22). (Right) The proportion of nontargeted iPSC clones from the same experiment with one or both DNMT3B alleles disrupted by NHEJ is also shown for wtCas9 (n = 25) and Cas9-Gem (n = 23). Data represent mean ± s.e.m. from three independent experiments. b–d adapted from ref. , Howden et al. Het, heterozygous; Hom, homozygous; NLS, nuclear localization signal.

Figure 2

Time line and overview of the one-step reprogramming/gene-editing workflow.

Figure 3

Generation of MAFP:mTagBFP2 reporter iPSC lines following one-step gene editing/reprogramming of healthy fibroblasts. (a) Schematic diagram of the MAFB locus and the homologous template used for reporter gene knock-in. The primers (ODNs) used for identifying targeted clones by PCR are indicated. (b) Representative gel following PCR analysis of 17 iPSC clones using primers ODN 1 and ODN 2, which flank the 3′ recombination junction. ODN 1 binds within the reporter cassette, and ODN 2 binds outside the region of homology. Arrows indicate PCR products amplified from correctly targeted clones. (c) PCR analysis of correctly targeted clones using primers ODN 3 and ODN 2, which flank the mTagBFP2 reporter cassette. These primers preferentially amplify the untargeted allele in heterozygous clones but will amplify only the knock-in alleles in homozygous clones. (d) Generation of kidney organoids from MAFB:mTagBFP2 iPSCs reveals appropriate mTagBFP2 reporter gene expression that is localized and restricted to the podocyte population in developing glomeruli. Scale bars, 500 µm. Ex, exon; pA, polyA signal.

Figure 4

Simultaneous reprogramming and genetic correction of fibroblasts from a kidney disease patient with an autosomal dominant mutation in HNF4A. (a) Schematic diagram of the HNF4A gene and the homologous template used for gene repair. The patient mutation (c.187C>T) (pink box), sgRNA, and 3-bp synonymous change (blue box) incorporated into the repair template and allele-specific primer used for identification of gene-corrected clones are shown. (b) Representative gel following PCR analysis of 18 iPSC clones using primers that specifically amplify the successfully gene-edited allele. Arrows indicate PCR products amplified from correctly edited clones. (c) Sanger sequencing analysis of the HNF4A target region of an uncorrected and a gene-corrected iPSC clone. *, patient-specific mutation; Ser, serine residue.

Figure 5

Generation of iPSC lines with a point mutation in H3F3A following one-step gene editing/reprogramming of healthy fibroblasts. (a) Schematic diagram of the H3F3A gene and the ssODN used for gene repair. The sgRNA, 3-bp synonymous change (blue box), and G > A mutation (pink box) incorporated into the ssODN are shown. The allele-specific primer used for identification of gene-corrected clones is also shown. (b) Representative gel following PCR analysis of 18 iPSC clones using primers that specifically amplify the successfully gene-edited allele. The arrow indicates PCR product amplified from a correctly edited clone. (c) Sequencing analysis of the H3F3A target region of a normal and two gene-edited (one heterozygous and one homozygous) iPSC clones. The 3-bp synonymous change introduced by the repair template is indicated.