Deepak Reyon

Efficient genome editing in zebrafish using a CRISPR-Cas system

In bacteria, foreign nucleic acids are silenced by clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems. Bacterial type II CRISPR systems have been adapted to create guide RNAs that direct site-specific DNA cleavage by the Cas9 endonuclease in cultured cells. Here we show that the CRISPR-Cas system functions in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies similar to those obtained using zinc finger nucleases and transcription activator–like effector nucleases.In bacteria, foreign nucleic acids are silenced by clustered, regularly interspaced, short palindromic repeats (CRISPR)--CRISPR-associated (Cas) systems. Bacterial type II CRISPR systems have been adapted to create guide RNAs that direct site-specific DNA cleavage by the Cas9 endonuclease in cultured cells. Here we show that the CRISPR-Cas system functions in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies similar to those obtained using zinc finger nucleases and transcription activator-like effector nucleases.

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.

Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.

Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) are highly efficient genome editing tools. CRISPR-associated 9 (Cas9) RGNs are directed to genomic loci by guide RNAs (gRNAs) containing 20 nucleotides that are complementary to a target DNA sequence. However, RGNs can induce mutations at sites that differ by as many as five nucleotides from the intended target. Here we report that truncated gRNAs, with shorter regions of target complementarity <20 nucleotides in length, can decrease undesired mutagenesis at some off-target sites by 5,000-fold or more without sacrificing on-target genome editing efficiencies. In addition, use of truncated gRNAs can further reduce off-target effects induced by pairs of Cas9 variants that nick DNA (paired nickases). Our results delineate a simple, effective strategy to improve the specificities of Cas9 nucleases or paired nickases.

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: n nMiller 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). n nWe 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. n nTo 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). n n n nFigure 1 n nTarget sequences, frequencies of mutations, and sequences of mutations induced by TALE nucleases in embryonic zebrafish cells n n n nTo 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). n nAn 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. n nIn 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.

Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins

Genome-wide studies have defined cell type–specific patterns of DNA methylation that are important for regulating gene expression in both normal development and disease. However, determining the functional significance of specific methylation events remains challenging, owing to the lack of methods for removing such modifications in a targeted manner. Here we describe an approach for efficient targeted demethylation of specific CpGs in human cells using fusions of engineered transcription activator–like effector (TALE) repeat arrays and the TET1 hydroxylase catalytic domain. Using these TALE-TET1 fusions, we demonstrate that modification of critical methylated promoter CpG positions can lead to substantial increases in the expression of endogenous human genes. Our results delineate a strategy for understanding the functional significance of specific CpG methylation marks in the context of endogenous gene loci and validate programmable DNA demethylation reagents with potential utility for research and therapeutic applications.Time-course evaluation of TALE-TET1-indcued effects on HBB promoter methylation and gene expression To test whether the -266 CpG demethylation and increased HBB gene expression induced by HB-4, HB-5-, and HB-6 are stable over time, we assayed these parameters in a more extended time-course experiment. K562 cells transfected with expression plasmids encoding HB-4, HB-5, HB-6, or a control TALE-TET1 fusion targeted to the KLF4 locus were assayed for -266 CpG demethylation and HBB gene expression at 4, 7, 14, and 30 days post-transfection. HB-4, HB5, and HB-6 fusions all showed their highest levels of fold-activation at post-transfection day 4 with HBB expression steadily decreasing by day 30 to the same level observed with the control KLF4-targeted TALE-TET1 protein (Supplementary Fig. 10a). Strikingly, for all three HBBtargeted TALE-TET1 proteins, the extent of -266 CpG methylation over time in the population of transfected cells directly paralleled the fold-activation of HBB gene expression while the methylation status of another CpG at position -306 did not change significantly during the course of the experiment (Supplementary Fig. 10b).

Locus-specific editing of histone modifications at endogenous enhancers using programmable TALE-LSD1 fusions

Mammalian gene regulation is dependent on tissue-specific enhancers that can act across large distances to influence transcriptional activity. Mapping experiments have identified hundreds of thousands of putative enhancers whose functionality is supported by cell type–specific chromatin signatures and striking enrichments for disease-associated sequence variants. However, these studies did not address the in vivo functions of the putative elements or their chromatin states and did not determine which genes, if any, a given enhancer regulates. Here we present a strategy to investigate endogenous regulatory elements by selectively altering their chromatin state using programmable reagents. Transcription activator–like (TAL) effector repeat domains fused to the LSD1 histone demethylase efficiently remove enhancer-associated chromatin modifications from target loci, without affecting control regions. We find that inactivation of enhancer chromatin by these fusion proteins frequently causes downregulation of proximal genes, revealing enhancer target genes. Our study demonstrates the potential of epigenome editing tools to characterize an important class of functional genomic elements.

High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases

We report here an efficient method for targeted mutagenesis of Arabidopsis genes through regulated expression of zinc finger nucleases (ZFNs)—enzymes engineered to create DNA double-strand breaks at specific target loci. ZFNs recognizing the Arabidopsis ADH1 and TT4 genes were made by Oligomerized Pool ENgineering (OPEN)—a publicly available, selection-based platform that yields high quality zinc finger arrays. The ADH1 and TT4 ZFNs were placed under control of an estrogen-inducible promoter and introduced into Arabidopsis plants by floral-dip transformation. Primary transgenic Arabidopsis seedlings induced to express the ADH1 or TT4 ZFNs exhibited somatic mutation frequencies of 7% or 16%, respectively. The induced mutations were typically insertions or deletions (1–142 bp) that were localized at the ZFN cleavage site and likely derived from imprecise repair of chromosome breaks by nonhomologous end-joining. Mutations were transmitted to the next generation for 69% of primary transgenics expressing the ADH1 ZFNs and 33% of transgenics expressing the TT4 ZFNs. Furthermore, ≈20% of the mutant-producing plants were homozygous for mutations at ADH1 or TT4, indicating that both alleles were disrupted. ADH1 and TT4 were chosen as targets for this study because of their selectable or screenable phenotypes (adh1, allyl alcohol resistance; tt4, lack of anthocyanins in the seed coat). However, the high frequency of observed ZFN-induced mutagenesis suggests that targeted mutations can readily be recovered by simply screening progeny of primary transgenic plants by PCR and DNA sequencing. Taken together, our results suggest that it should now be possible to obtain mutations in any Arabidopsis target gene regardless of its mutant phenotype.