Decades of work have aimed to genetically reprogram T cells for therapeutic purposes using recombinant viral vectors, which do not target transgenes to specific genomic sites. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

BackgroundConditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient.ResultsHere we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5–100% of the resulting live offspring.Conclusions Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1220-4) contains supplementary material, which is available to authorized users.

CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1-10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts-CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30-60% and reaches as high as 100% in some cases). The protocol takes ∼2 months to generate the founder mice.

The availability of techniques to create desired genetic mutations has enabled the laboratory mouse as an extensively used model organism in biomedical research including human genetics. A new addition to this existing technical repertoire is the CRISPR/Cas system. Specifically, this system allows editing of the mouse genome much faster than the previously used techniques and more importantly multiple mutations can be created in a single experiment. Here we provide protocols for preparation of CRISPR/Cas reagents and microinjection into one cell mouse embryos to create knockout or knock-in mouse models.

The major barrier to effective non-viral T cell genome targeting of large DNA sequences has been the toxicity of the DNA 10 . While the introduction of short singlestranded oligodeoxynucleotide (ssODN) HDR templates does not cause significant T cell death, it has been shown that larger linear double stranded (dsDNA) templates are toxic at high concentrations 11,12 . Contrary to expectations, we found that co-electroporation of human primary T cells with CRISPR-Cas9 ribonucleoprotein (Cas9 RNP 13,14 ) complexes and long (>1kb) linear dsDNA templates reduced the toxicity associated with the dsDNA template (Extended Data Fig 1). Cas9 RNPs were co-electroporated with a dsDNA HDR template designed to introduce an N-terminal GFP-fusion in the housekeeping peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/183418 doi: bioRxiv preprint first posted online Aug. 31, 2017; 3 gene RAB11A (Fig. 1a). Systematic exploration of this approach while optimizing for both viability and efficiency ( Fig. 1b and Extended Data Fig. 2) resulted in GFP expression in ~50% of cells in both primary human CD4+ and CD8+ T cells. The method was reproducibly efficient while maintaining high cell viability and expandability (Fig. 1c, d, e, and Extended Data Fig. 3). The system is also compatible with current manufacturing protocols for cell therapies as it could be applied to fresh or cryopreserved cells, bulk T cells or FACS-sorted sub-populations, and cells from whole blood or leukapheresis (Extended Data Fig. 4).We next confirmed that the system could be applied broadly by targeting sequences in different locations throughout the genome. We efficiently engineered GFP+ primary T cells by generating fusions with different genes (Fig. 2a and Fig. 3a and Extended Data Fig. 14). One mutation, c.530A>G, creates a premature stop codon. With non-viral genome targeting, we were able to correct the mutation and observe IL2RA expression on the surface of corrected T cells from the patient (Fig. 3b). Long dsDNA templates led to efficient correction of the mutations. Because only two base pair changes were necessary (one to correct the mutation and one to silently remove the gRNA's PAM sequence), a short single-stranded DNA (~120 bps) could also be used to make the correction. These single-stranded DNAs were able to correct the mutation at high frequencies, although the efficiency of correction was lower than with the longer dsDNA template (Extended Data Fig. 15, 16).Correction was successful in T cells from all three siblings, but lower rates of IL2RA expression were seen in compound het 3, which could be due to altered cell-state associated with the patient's disease or the fact she was the only sibling treated with immunosuppressive therapy (Extended Data Table 1 and Extended Data Fig. 17). The second mutation identified, c.800delA, causes a frameshift in the reading frame of the final IL2RA exon. This frameshift mutation c...