New Human Chromosomal Sites with "Safe Harbor" Potential for Targeted Transgene Insertion

Abstract

This study identified 35 new sites for targeted transgene insertion that have the potential to serve as new human genomic "safe harbor" sites (SHS). SHS potential for these 35 sites, located on 16 chromosomes, including both arms of the human X chromosome, and for the existing human SHS AAVS1, hROSA26, and CCR5 was assessed using eight different desirable, widely accepted criteria for SHS verifiable with human genomic data. Three representative newly identified sites on human chromosomes 2 and 4 were then experimentally validated by in vitro and in vivo cleavage-sensitivity tests, and analyzed for population-level and cell line-specific sequence variants that might confound site targeting. The highly ranked site on chromosome 4 (SHS231) was further characterized by targeted homology-dependent and -independent transgene insertion and expression in different human cell lines. The structure and fidelity of transgene insertions at this site were confirmed, together with analyses that demonstrated stable expression and function of transgene-encoded proteins, including fluorescent protein markers, selectable marker cassettes, and Cas9 protein variants. SHS-integrated transgene-encoded Cas9 proteins were shown to be capable of introducing a large (17 kb) gRNA-specified deletion in the PAX3/FOXO1 fusion oncogene in human rhabdomyosarcoma cells and as a Cas9-VPR fusion protein to upregulate expression of the muscle-specific transcription factor MYF5 in human rhabdomyosarcoma cells. An engineering "toolkit" was developed to enable easy use of the most extensively characterized of these new human sites, SHS231, located on the proximal long arm of chromosome 4. The target sites identified here have the potential to serve as additional human SHS to enable basic and clinical gene editing and genome-engineering applications.

Keywords: Cas9 nucleases; gene therapy; genome editing; human genome; transgene insertion site; “safe harbor” site.

Figure 1.

Identification and mapping of new human “safe harbor” sites (SHS). (A) The canonical mCreI homing endonuclease cleavage site is shown at the top, with twofold symmetric base-pair positions shaded. Shown below, the target site is a position weight matrix (PWM; also often referred to as a position-specific site matrix [PSSM]) that summarizes biochemical data on the functional consequences of each possible base substitution on cleavage sensitivity at each mCreI target site position, scaled so that a value of 1 = native site cleavage sensitivity and values <0.3 indicate cleavage resistance. Base pairs highlighted in yellow indicate either the canonical base pair at that position or a highly cleavable base-pair substitution. (B) Work flow that uses these PWN/PSSM data to initiate the search for and evaluation of predicted highly cleavage-sensitive mCreI target sites in the human genome. (C) Physical confirmation and functional verification of two new unique SHS located on chromosomes 2p (SHS229) and 4q (SHS231). A third highly ranked SHS (SHS253) was identified at six locations on the short arms of chromosomes 2, 5, and X and on the long arms of chromosomes 7, 14, and 17. Asterisks indicate sites where base-pair variants have been identified in the mCreI target site in human population genetic data (see text for additional details).

Figure 2.

Structure of three representative new target sites indicating location of mCreI, Cas9, and TALEN target sites. The top two sequence diagrams detail features of the chr2 SHS229 and SHS253, whereas the bottom diagram provides additional detail and results on the chr 4q SHS231. Locations of cleavage sites for mCreI, TALEN, and CRISPR/Cas9 nucleases centered on mCreI target/cleavage sites (key shown top right). The SHS231 repair template is shown below the chr 4 target site region, which also indicates the location of the variable 108 bp SINE-derived insertion sequence identified in one or both alleles of a subset of the cell lines examined. The bottom panel shows independently cloned and sequenced inserts from targeted SHS231 insertions by all three nucleases. The mCreI targeting experiments used an expression vector that encoded both mCreI and the TREX2 nuclease (see text), and Cas9 targeting was performed using a common guide RNA and either a Cas9 cleavase or nickase. Numbers to the right of each row indicate the number of independent targeting events that were cloned and sequenced (see text for additional details).

Figure 3.

Integration of transgenes into chromosome 4q SHS231 locus using homology-independent non-homologous end joining (NHEJ) DNA repair mechanisms. (A) Homology-independent transgene integration is mediated by targeting both the repair template and genomic target site using dual CRISPR guide RNAs (gRNAs; blue and green triangles represent gRNA target sites). The structure of a SH231 repair template expressing the puromycin resistance gene is indicated, including the size of a puromycin transgene cassette and locations of CRISPR gRNAs. Representative sequences from the 5′ transgene integration site after knock-in-specific polymerase chain reaction (PCR) amplification (PCR primers; purple arrows). (B) Relative knock-in efficiency of a puromycin cassette at the SHS231 locus using homology-independent repair (US2-Cas9; NHEJ) and homology-directed repair (nCas9, Cas9, mCreI; HDR) compared to random piggybac transposition (PBase) in the rhabdomyosarcoma (RMS) cell lines RD and SMS-CTR. Crystal violet staining was used to visualize the percentage of stable puromycin-resistant colonies resulting from 3 × 10⁴ transfected cells after 10 days in culture. No colonies were identified from CRISPR only or repair template only controls. (C) Quantification of crystal violet staining from SHS231 knock-in stable cells generated from the same RMS cell lines used in (B) above. Asterisk indicates significant difference between NHEJ- and HDR-mediated SHS231 knock-in approaches, p < 0.05.

Figure 4.

Stable expression, functional gene editing, and gene activation by SHS231 integrated transgenes. (A) Long-term stable green fluorescent protein (GFP) expression from a SHS231 integrated transgene in two independent RMS cell lines Rh5 and SMS-CTR cultured in the absence of antibiotic selection. (B) Relative Cas9 expression level (cycle threshold [Ct]) from a SHS231 integrated Cas9 cassette compared to cells transduced with high-titer Cas9 expressing lentivirus or the endogenous expression level of GAPDH. Both SHS231 and lentiviral Cas9 variants were expressed from the human EF1α promoter. (C) Targeted deletion of a 17,188 bp gDNA segment of the PAX3/FOXO1 fusion oncogene in a third RMS cell line, Rh30, by Cas9 protein expressed from the SHS231 locus. Dual gRNA target sites (blue and green triangles) and deletion PCR primer sites (purple arrows) are identified. (D) Demonstration of endogenous MYF5 gene activation with SHS231 expressed dCas9-VPR and Cas9-VPR transgenes relative to wild-type (unmodified) RMS cells. Gene activation was achieved by targeting full-length (20 bp) or truncated (14 bp) gRNAs (blue, green, and red triangles) to the promoter region of the MYF5 gene.