Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells

Abstract

Sequence-specific nucleases such as TALEN and the CRISPR/Cas9 system have so far been used to disrupt, correct or insert transgenes at precise locations in mammalian genomes. We demonstrate efficient 'knock-in' targeted replacement of multi-kilobase genes in human induced pluripotent stem cells (iPSC). Using a model system replacing endogenous human genes with their mouse counterpart, we performed a comprehensive study of targeting vector design parameters for homologous recombination. A 2.7 kilobase (kb) homozygous gene replacement was achieved in up to 11% of iPSC without selection. The optimal homology arm length was around 2 kb, with homology length being especially critical on the arm not adjacent to the cut site. Homologous sequence inside the cut sites was detrimental to targeting efficiency, consistent with a synthesis-dependent strand annealing (SDSA) mechanism. Using two nuclease sites, we observed a high degree of gene excisions and inversions, which sometimes occurred more frequently than indel mutations. While homozygous deletions of 86 kb were achieved with up to 8% frequency, deletion frequencies were not solely a function of nuclease activity and deletion size. Our results analyzing the optimal parameters for targeting vector design will inform future gene targeting efforts involving multi-kilobase gene segments, particularly in human iPSC.

Figure 1.

Homozygous targeted gene replacement using one or two CRISPR sgRNAs. (A) Two Crispr sgRNAs target hThy1 within intron 1 (L1) or after the polyadenylation sites (R1). The mThy1 targeting vector plasmid contains mThy1 exons 2 and 3 (orange), flanked by hThy1 homology arms outside the sgRNA sites—coding exon 1 (which encodes the signal peptide) is retained but the sgRNA sites are disrupted. Small triangles indicate the primer sites for the four genotyping PCR reactions. (B) PGP1 iPSC were nucleofected with plasmids encoding the mThy1 targeting vector, the Cas9 nuclease, and L1, R1, both, or no sgRNAs. Five days later, cells were analyzed by flow cytometry. The percentage of cells that have gained mThy1 expression and/or lost hThy1 expression are indicated. (C) Single iPSC were FACS sorted from each quadrant, cultured in individual wells, and genotyped using the four PCR reactions. Alleles were identified based on the size and Sanger sequencing of the PCR products: native human (+); recombined mouse (m); excised between the two sgRNA sites (Δ); and inverted between the two sgRNA sites (i). Representative gels from +/+ wild type, m/+ heterozygous, m/m homozygous, m/Δ heterozygous, m/i heterozygous, Δ/Δ homozygous, and i/Δ heterozygous colonies are shown. (D) Frequency of genotypes among FACS-sorted iPSC colonies. Results are representative of three independent experiments.

Figure 2.

Targeted gene replacement with homology on either site of each cut site. (A) The mThy1 targeting vector from Figure 1 (outside) was modified such that the human Thy1 homology arms extend inside the L1 and R1 sgRNA sites. While mouse Thy1 exons 2 and 3 (orange) are completely retained in this targeting vector, 350 bp of mouse Thy1 intron 1 and 150 bp of mouse Thy1 sequence after the polyadenylation site was replaced with the corresponding human sequence. The resulting targeting vector contains intact L1 and R1 sgRNA sites (Intact). Next, a single base pair was deleted from each sgRNA site in the targeting vector to develop a alternate version with similar homology arms but disrupted sgRNA sites (Disrupted). (B and C) PGP1 or PGP4 iPSC were nucleofected with one of the mouse Thy1 targeting vectors (outside, Intact, or disrupted) along with plasmids encoding the Cas9 nuclease, and L1, R1, both, or no sgRNAs. (B) Two days post-nucleofection, the viable cells in each condition were counted. Viable cell counts were normalized to that of the Outside mouse Thy1 targeting vector with no sgRNA (100). Error bars show mean ± S.E.M. of at least three independent experiments. (C) Five days post-nucleofection, cells were analyzed by flow cytometry. The percentage of cells that have gained expression of mouse Thy1 and / or lost expression of human Thy1 are indicated. Results are representative of three (PGP1) and two (PGP4) independent experiments.

Figure 3.

Frequency of CRISPR-generated homozygous and heterozygous deletions. (A) Crispr sgRNAs were generated targeting the human Thy1 gene: two within intron 1 (left: L1 and L2), and 10 at various distances after hThy1 (right: R1 through R10). (B and C) Pairs of one left and one right sgRNA were nucleofected into either (B) PGP1 iPSC or (C) a Thy1^m/+ PGP1 iPSC clone. As a negative control, only a left sgRNA was nucleofected (right column). Five days later, cells were analyzed by flow cytometry for either (B) homozygous deletion of both human Thy1 alleles or (C) heterozygous deletion of the remaining human Thy1 allele and retention of the mouse Thy1 allele. The distance between the sgRNA sites (Thy1 Δ) and the frequency of hThy1⁻ cells is indicated. (D and E) The percentage of hThy1⁻ cells from each sgRNA pair minus that from the left sgRNA-only control is plotted against the size of the Thy1 deletion. sgRNA pairs that included L1 or L2 are shown in black or red, respectively. Error bars show mean ± S.E.M. of two independent experiments.