Cyd Khayter

High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells

Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell–based reporter assay to characterize off-target cleavage of CRISPR-associated (Cas)9-based RGNs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We also readily detected off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGNs can be highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.

FLASH assembly of TALENs for high-throughput genome editing

Engineered transcription activator–like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published, and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the fast ligation-based automatable solid-phase high-throughput (FLASH) system, a rapid and cost-effective method for large-scale assembly of TALENs. We tested 48 FLASH-assembled TALEN pairs in a human cell–based EGFP reporter system and found that all 48 possessed efficient gene-modification activities. We also used FLASH to assemble TALENs for 96 endogenous human genes implicated in cancer and/or epigenetic regulation and found that 84 pairs were able to efficiently introduce targeted alterations. Our results establish the robustness of TALEN technology and demonstrate that FLASH facilitates high-throughput genome editing at a scale not currently possible with other genome modification technologies.

Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing

Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI nucleases (RFNs) that can recognize extended sequences and edit endogenous genes with high efficiencies in human cells. RFN cleavage activity depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation substantially reducing the likelihood that a suitable target site will occur more than once in the genome and therefore improving specificities relative to wild-type Cas9 monomers. RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5′ end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing.

Targeted gene disruption in somatic zebrafish cells using engineered TALENs.

To the Editor: Miller et al. recently described a TALE nuclease architecture for performing efficient genome editing1. The authors demonstrated that TALE nucleases, composed of an engineered array of TALE repeats fused to the non-specific FokI cleavage domain, could be used to introduce targeted double-stranded breaks (DSBs) in human cells with high efficiency. Repair of these DSBs by normal DNA repair mechanisms such as non-homologous end-joining (NHEJ) or homologous recombination (HR) enables introduction of alterations at or near the site of the break. A single 34 amino acid TALE repeat binds to one bp of DNA and repeats that bind each of the four DNA bases have been described2, 3. These modules can be assembled into arrays capable of binding extended DNA sequences. TALE nucleases may have advantages over engineered zinc finger nucleases (ZFNs) due to the relative ease with which they can be designed and their potential ability to be targeted to a wide range of sequences (with target sites reported to be as frequent as 1 in 35 bps of random DNA sequence4). We sought to determine whether the TALE nuclease framework described by Miller et al. could also be used to efficiently modify endogenous genes in zebrafish. Previous studies have shown that error-prone repair of ZFN-induced DSBs by NHEJ can result in the efficient introduction of small insertions or deletions (indels) at cleavage sites in endogenous zebrafish genes5–7. These indels frequently result in frameshift knockout mutations that can be passed through the germline to create mutant fish5–9. ZFN technology has enabled reverse genetics studies to be performed in zebrafish, a capability that did not previously exist. However, engineering ZFNs can be challenging due to the need to account for context-dependent effects among individual fingers in an array. In addition, although many zebrafish genes can be targeted with ZFNs made by publicly available methods that account for context-dependence7, 10, it can in some instances be difficult to target within some genes in zebrafish due to the currently limited targeting range of publicly available ZFN engineering platforms. Thus, if TALE nucleases could be used to introduce targeted mutations in zebrafish, this platform would provide an important additional capability for this model organism. To test the ability of TALE nucleases to function in zebrafish, we targeted DNA sequences in two endogenous zebrafish genes gria3a and hey2 (Figure 1). To avoid confounding effects that might affect binding and cleavage of DNA sites by TALE nucleases (e.g.--chromatin structure or DNA methylation), we chose to target sequences that we had efficiently altered previously in zebrafish using engineered ZFNs (Supplementary Figures 1 and 2). Using an iterative assembly approach (Supplementary Methods), we constructed four TALE nuclease monomers to partially overlapping sites in the gria3a gene and two TALE nuclease monomers to a site in the hey2 gene (Figure 1 and Supplementary Figure 3). These six TALE nuclease monomers all harbor the wild-type FokI cleavage domain (Supplementary Figures 4 and 5) and can be paired in combinations to make three TALE nuclease dimers to the gria3a gene and one TALE nuclease dimer to the hey2 gene (Figure 1). We injected RNAs encoding the various TALE nuclease pairs into one-cell stage zebrafish embryos and determined the frequency of NHEJ-mediated mutagenesis at the target site by sequence analysis of alleles from pooled injected embryos (Supplementary Methods, Supplementary Figs. 6–10 and Supplementary Table 1). As shown in Figure 1, we found that all four pairs of TALE nucleases induced targeted indels with high mutation frequencies ranging from 11 to 33%. These frequencies are comparable to what we obtained with ZFNs targeted to DNA sequences in the same vicinity of the gene (Supplementary Figure 1); however, we note that TALE nucleases harbor wild-type FokI domains whereas the ZFNs harbor obligate heterodimeric FokI domains11. Although small indels were typically observed with the TALE nucleases, some large deletions (up to 303 basepairs) were also found (Figure 1). Figure 1 Target sequences, frequencies of mutations, and sequences of mutations induced by TALE nucleases in embryonic zebrafish cells To assess the toxicity of our engineered TALE nucleases, we scored the percentages of dead and deformed embryos that resulted from mRNA microinjections (Supplementary Figure 11). Although we cannot directly compare these results with the microinjections of ZFNs due to the differences between the FokI endonuclease domains used (EL/KK heterodimeric FokI11 for ZFNs versus wild-type FokI for the TALE nucleases) and the specific sequences targeted, the toxicity we observed with injection of 600 pg of TALE nuclease mRNAs (ranging between 40–80%) appears similar to that observed with 400–500 pg of mRNAs encoding ZFNs targeted to sequences in the same vicinity and to other genes (Supplementary Figure 12 and Reference 7). An important future experiment will be to demonstrate germline transmission of TALE nuclease-induced mutations. Given that the frequencies of mutation and the extent of toxicities we observe are similar to what we have seen with ZFNs, we expect that TALE nuclease-induced mutations should be efficiently passed through the germline to progeny and we are currently conducting experiments to test this prediction. Successful germline transmission of these mutations will be critical for using TALE nucleases to perform reverse genetics in zebrafish. Progeny fish bearing TALE nuclease-induced mutations, unlike founder F0 fish, will not be mosaic (i.e.--these fish will have uniform mutation of all cells in the organism); such mutant fish will enable determination of whether both mono-allelic and bi-allelic alterations of a gene are possible and will provide a more straightforward background on which to perform analysis of off-target effects. In summary, we show that the TALE nuclease framework described by Miller et al. can be used to efficiently introduce targeted indel mutations in endogenous zebrafish genes at the somatic cell level. Although in this study we chose two genomic loci that have been successfully targeted with ZFNs before, all six TALE nuclease monomers we constructed showed high mutagenesis activities when tested in various pairwise combinations, suggesting that the TALE nuclease framework is also highly robust and effective in zebrafish. As is the case with ZFNs, the complete genome-wide spectrum of off-target mutations introduced by TALE nucleases remains unknown. However, expression of the TALE nucleases we made in zebrafish does not show toxicity substantially different from that observed with expression of ZFNs, suggesting that the magnitude of off-target effects may be comparable with the two types of nucleases. In principle, off-target mutations made by TALE nucleases can be removed by out-crossing the founder assuming that they are not tightly linked to the intended mutation. In addition, mutant phenotypes could also be confirmed by generation of a second mutant allele using nucleases targeted to a different site. For mutagenesis of genes in zebrafish (and other model organisms such as C. elegans12), TALE nucleases may offer potential advantages over ZFNs because they can be easily and quickly assembled in a modular fashion and they can potentially target a greater range of DNA sequences. Thus, we expect that the ability to utilize both ZFNs and TALE nucleases should enable any researcher to rapidly and easily create targeted mutations in their endogenous zebrafish gene of interest.

Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA)

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe context-dependent assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA-generated ZFNs, we rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multigene pathways or genome-wide alterations.

In Situ Genetic Correction of the Sickle Cell Anemia Mutation in Human Induced Pluripotent Stem Cells Using Engineered Zinc Finger Nucleases

The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient‐derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene‐corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug‐resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN‐induced break. Our approach delineates a roadmap for using ZFNs made by an open‐source method to achieve efficient, transgene‐free correction of monogenic disease mutations in patient‐derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene‐corrected human iPSCs that could be used for therapeutic applications. STEM CELLS 2011;29:1717–1726

Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs

Transcription activator-like effector nucleases (TALENs) are powerful new research tools that enable targeted gene disruption in a wide variety of model organisms. Recent work has shown that TALENs can induce mutations in endogenous zebrafish genes, but to date only four genes have been altered, and larger-scale tests of the success rate, mutation efficiencies and germline transmission rates have not been described. Here, we constructed homodimeric TALENs to 10 different targets in various endogenous zebrafish genes and found that 7 nuclease pairs induced targeted indel mutations with high efficiencies ranging from 2 to 76%. We also tested obligate heterodimeric TALENs and found that these nucleases induce mutations with comparable or higher frequencies and have better toxicity profiles than their homodimeric counterparts. Importantly, mutations induced by both homodimeric and heterodimeric TALENs are passed efficiently through the germline, in some cases reaching 100% transmission. For one target gene sequence, we observed substantially reduced mutagenesis efficiency for a variant site bearing two mismatched nucleotides, raising the possibility that TALENs might be used to perform allele-specific gene disruption. Our results suggest that construction of one to two heterodimeric TALEN pairs for any given gene will, in most cases, enable researchers to rapidly generate knockout zebrafish.

Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs)

Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%–76.8% compared to 1.1%–3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

Engineering Designer Transcription Activator‐‐Like Effector Nucleases (TALENs) by REAL or REAL‐Fast Assembly

Engineered transcription activator–like effector nucleases (TALENs) are broadly useful tools for performing targeted genome editing in a wide variety of organisms and cell types including plants, zebrafish, C. elegans, rat, human somatic cells, and human pluripotent stem cells. Here we describe detailed protocols for the serial, hierarchical assembly of TALENs that require neither PCR nor specialized multi‐fragment ligations and that can be implemented by any laboratory. These restriction enzyme and ligation (REAL)–based protocols can be practiced using plasmid libraries and user‐friendly, Web‐based software that both identifies target sites in sequences of interest and generates printable graphical guides that facilitate assembly of TALENs. With the described platform of reagents, protocols, and software, researchers can easily engineer multiple TALENs within 2 weeks using standard cloning techniques. Curr. Protoc. Mol. Biol. 100:12.15.1‐12.15.14.

Evaluation of OPEN Zinc Finger Nucleases for Direct Gene Targeting of the ROSA26 Locus in Mouse Embryos

Zinc finger nucleases (ZFNs) enable precise genome modification in a variety of organisms and cell types. Commercial ZFNs were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. Here we report precise targeted mutagenesis of the mouse genome using Oligomerized Pool Engineering (OPEN) ZFNs. OPEN ZFN can be constructed using publicly available resources and therefore provide an attractive alternative for academic researchers. Two ZFN pairs specific to the mouse genomic locus gt(ROSA26)Sor were generated by OPEN selections and used for gene disruption and homology-mediated gene replacement in single cell mouse embryos. One specific ZFN pair facilitated non-homologous end joining (NHEJ)-mediated gene disruption when expressed in mouse zygotes. We also observed a single homologous recombination (HR)-driven gene replacement event when this ZFN pair was co-injected with a targeting vector. Our experiments demonstrate the feasibility of achieving both gene ablation through NHEJ and gene replacement by HR by using the OPEN ZFN technology directly in mouse zygotes.