Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

/pdf/multiplex-genome-engineering-using-crispr-cas-systems-2qm4sjme5b.pdf

Multiplex Genome Engineering Using CRISPR/Cas Systems

Genome Editing Clustered regularly interspaced short palindromic repeats (CRISPR) function as part of an adaptive immune system in a range of prokaryotes: Invading phage and plasmid DNA is targeted for cleavage by complementary CRISPR RNAs (crRNAs) bound to a CRISPR-associated endonuclease (see the Perspective by van der Oost). Cong et al. (p. 819, published online 3 January) and Mali et al. (p. 823, published online 3 January) adapted this defense system to function as a genome editing tool in eukaryotic cells. A bacterial genome defense system is adapted to function as a genome-editing tool in mammalian cells. [Also see Perspective by van der Oost] Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.

/pdf/genome-engineering-using-the-crispr-cas9-system-4x8nw03mgu.pdf

Genome engineering using the CRISPR-Cas9 system

The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

The new frontier of genome engineering with CRISPR-Cas9

http://knockout.cwru.edu/publications/hsucrisprreview.pdf

Development and applications of CRISPR-Cas9 for genome engineering.

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.

/pdf/high-frequency-off-target-mutagenesis-induced-by-crispr-cas-298nfv8p0t.pdf

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

CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide, off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint 'hotspots'. Finally, GUIDE-seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced, off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases before clinical use.

/pdf/guide-seq-enables-genome-wide-profiling-of-off-target-45karz7nsv.pdf

GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases

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.

/pdf/flash-assembly-of-talens-for-high-throughput-genome-editing-2u6q96loq4.pdf

FLASH assembly of TALENs for high-throughput 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 recognize extended sequences and can edit endogenous genes with high efficiencies in human cells. The cleavage activity of an RFN depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation and therefore show improved specificities relative to wild-type Cas9 monomers. Importantly, direct comparisons show that 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.

/pdf/dimeric-crispr-rna-guided-foki-nucleases-for-highly-specific-4t2ux5esdf.pdf

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

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.

Cyd Khayter

Papers

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

GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases

FLASH assembly of TALENs for high-throughput genome editing

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

Targeted gene disruption in somatic zebrafish cells using engineered TALENs.