Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification

Abstract

In T cells with transgenic high-avidity T cell receptors (TCRs), endogenous and transferred TCR chains compete for surface expression and may pair inappropriately, potentially causing autoimmunity. To knock out endogenous TCR expression, we assembled 12 transcription activator-like effector nucleases (TALENs) and five guide RNAs (gRNAs) from the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system. Using TALEN mRNA, TCR knockout was successful in up to 81% of T cells. Additionally, we were able to verify targeted gene addition of a GFP gene by homology-directed repair at the TALEN target site, using a donor suitable for replacement of the reporter transgene with therapeutic TCR chains. Remarkably, analysis of TALEN and CRISPR/Cas9 specificity using integrase-defective lentiviral vector capture revealed only one off-target site for one of the gRNAs and three off-target sites for both of the TALENs, indicating a high level of specificity. Collectively, our work shows highly efficient and specific nucleases for T cell engineering.

Keywords: T cell therapy; gene editing; off target.

Graphical abstract

Figure 1

DSB Repair and Targeted Genome Editing of the TCR Loci Using Designer Nucleases (A) During NHEJ-repair of TALEN- and CRISPR/Cas9-induced DSBs, frameshift mutations can result in gene knockout, or episomal IDLV can be integrated into DSBs, allowing for the permanent marking of off-target DSBs. If donor DNA is provided, HDR can lead to targeted integration of an expression cassette, i.e., therapeutic TCR chains. (B) The TCR α and β locus are composed of a number of variable (V), joining (J), constant (C), and, in the case of TRBC, diversity (D) gene segments (numbers of functional genes from IMGT/GENE-DB version 3.1.16, December 14, 2016). The positions of TALEN and gRNA target sequences for TCR knockout in the TCR α constant region (TRAC) and a homologous sequence shared by both TCR β constant regions (TRBC1/2) are marked by scissor symbols. DSB, DNA double-strand break; gRNA, guide RNA; HDR, homology-directed repair; IDLV, integrase-defective lentiviral vector; LTR, long terminal repeat; NHEJ, non-homologous end joining; PAM, protospacer adjacent motif; RVD, repeat variable di-residue.

Figure 2

Evaluation of TALEN and CRISPR/Cas9 Activity (A) Positions of TALEN and CRISPR/Cas9 gRNA binding sites at the TRAC target site. (B) TALEN and CRISPR/Cas9 activity in the TRAC locus in K562 cells. (C) Activity of obligate heterodimeric TALENs in the TRAC locus in 293T cells. (D) TALEN and gRNA binding sites at TRBC1 and TRBC2 target locus. (E) TALEN activity in the TRBC1 and TRBC2 locus in 293T cells. (F) CRISPR/Cas9 activity in the TRBC1 and TRBC2 locus in K562 cells. (B, C, E, and F) PCR amplification of the target regions in the TCR loci produces upper bands. T7EI-mediated cleavage of NHEJ-originated heteroduplex DNA results in additional cleavage bands, marked by arrowheads. A SNP in the TRBC2 locus results in additional bands, marked by arrows (>). Ctrl, negative control; M, marker; Sp., length of spacer between TALEN binding sites in base pairs; TALEN_G, GoldyTALEN; TALEN_P(OH), pTAL3 (obligate heterodimeric FokI domain); TALEN_S, CAG-T7-TALEN(Sangamo)-Destination.

Figure 3

Analysis of CRISPR/Cas9 and TALEN-Mediated TCR Knockout in Primary T Cells (A) Percentage of TCR knockout, evaluated by flow cytometric analysis of cell surface CD3 expression, in T cells 6 days after electroporation with αC2 or βC2. Shown are representative flow cytometry plots from four independent experiments with duplicates in T cells from three different donors. (B) Confirmation of CRISPR/Cas9 activity in T cells by T7EI assay and deep sequencing. (C) Percentage of TCR knockout, quantified by FACS, in T cells 6 days after electroporation with αT4_m or βT4_m. Shown are representative flow cytometry analyses of six independent experiments, with duplicates, in T cells from five different donors. (D) TALEN activity at target sites validated by T7EI assay and deep sequencing after electroporation of primary T cells with αT4_m or βT4_m. (B and D) Upper bands derived from wild-type PCR products and lower T7EI cleavage products are marked by arrowheads. A SNP in the TRBC1 locus results in an additional band, marked by arrows (>). The frequency of indels was analyzed by deep sequencing of PCR products. Ctrl, untreated T cells as negative control; FSC, forward scatter; M, marker; n.d., not determined.

Figure 4

Quantification of NHEJ at TALEN and CRISPR/Cas9 On- and Off-Target Sites, Analyzed by Deep Sequencing and the CRISPResso Tool Percentage of sequences showing indels (combined insertions and deletions) at each position of the respective on- and off-target site amplicon in αT4_m- and βT4_m-treated primary T cells and βC1-treated K562 cells. Only indels overlapping the specified window of 20 bp that is centered on the respective predicted cleavage site are included in the quantification indicated in each graph. MM, number of mismatches to the respective target site; NC, non-coding.

Figure 5

TALEN-Induced, HDR-Mediated, GFP Integration into the TRAC Locus in K562 Cells and Primary T Cells (A) Percentage of GFP-expressing K562 cells 14 days after nucleofection with GFP-encoding donor plasmid TA200G or TA800G without (upper panel) or together with TALEN αT4_P or βT4_P (lower panel). Representative FACS analyses of two independent experiments are shown. (B) Validation of targeted integration into the TRAC locus by bidirectional PCR with primers binding in the GFP expression cassette or outside of the 5′ or 3′ homology arms, respectively. (C) Flow cytometric analysis of TCR knockout, determined by CD3 surface expression, and GFP expression of primary T cells 7, 15, and 21 days after transduction with TA800G-IDLV only (right panels) or additional electroporation with αT4_m on day 1 (left panels). Shown are representative FACS analyses of three independent experiments in T cells from separate donors. (D) Validation of targeted integration into the TRAC locus by bidirectional PCR with primers binding in the GFP expression cassette or outside of the 5′ or 3′ homology arm, respectively (see scheme in B). PCR products are marked by arrowheads. Hom., homology region.