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

CRISPR RNA-guided endonucleases (RGENs) 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 Cas9-based RGENs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We readily detected off-target alterations induced by four out of six RGENs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbor up to five mismatches and many are mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGENs are highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications

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 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

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

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 ZFNs, we rapidly altered 20 genes in zebrafish, Arabidopsis, and soybean. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multi-gene pathways or genome-wide alterations

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

TALENs and ZFNs exhibit similar toxicity profiles.

<p>The cumulative percentage of embryos that were dead or deformed by 3 days post-fertilization is denoted. CoDA ZFNs from the same genes are shown on the right. The number of fish examined for toxicity is shown below; the mutation rate is derived from a pool of 12 embryos.</p

Non-homologous end joining repair of ZFN-generated double-strand breaks within the <i>ROSA26</i> locus.

<p>(A) Schematic of ZFN 90/91 and 204/205 target sites within <i>ROSA26</i> intron 1. ZFN pairs 90/91 and 204/205 target sites 75 bp and 403 bp upstream of the <i>Xba</i>I site (white arrows), which is routinely used in <i>ROSA26</i> targeting, respectively. ZFNs 204/205 target a partial <i>Fsp</i>I recognition sequence. RF and RR, <i>ROSA26</i> forward and reverse primers used for NHEJ analysis generating a 474 bp fragment (black arrows). (B) Screening for NHEJ repair at the ZFN204/205 cleavage site. Genomic DNA extracted from fetuses or pups developing from ZFN-injected zygotes was amplified with primers RF and RR and subjected to <i>Fsp</i>I restriction digest. Most error-prone NHEJ repair events eliminate the <i>Fsp</i>I recognition sequence (underlined in C) resulting in an indigestible band at 474 bp. In the majority of founders such as Z20 both modified and wt alleles were detected, however only mutated alleles were present in founder ZGFP112. (C) Cloning and sequencing of undigested PCR products reveals mutations around the ZFN204/205 cleavage site. Founder ZGFP112 carried an identical Δ23 deletion in both <i>ROSA26</i> alleles. ZFN 204/205 recognition sites highlighted in bold and the spacer region in grey color.</p

Genome engineering using ZFNs and TALENs.

<p>ZFNs utilize DNA binding domains that recognize ∼3 bp sequences and are joined together to create arrays that can target specific DNA sequences. TALENs bind DNA using TAL effector repeat domains derived from <i>Xanthomonas</i> that recognize individual nucleotides. These TALE repeats are ligated together to create binding arrays that recognize extended DNA sequences. Each ZFN or TALEN binds to a half-site with dimeric FokI nuclease domains cleaving the DNA within the intervening spacer region. The mechanism responsible for inducing DNA mutations is identical using either methodology, where nuclease-induced double stranded DNA breaks are repaired by error-prone non-homologous end joining (NHEJ) resulting in the creation of insertion or deletion mutations (indels).</p