A Co-CRISPR Strategy for Efficient Genome Editing in Caenorhabditis elegans

Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.     

Genome Editing—Principles and Applications for Functional Genomics Research and Crop Improvement

Genome editing technologies are powerful tools for studying gene function and for crop improvement. The technologies rely on engineered endonucleases to generate double stranded breaks (DSBs) at target loci. The DSBs are repaired through the error-prone non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways in cells, resulting in mutations and sequence replacement, respectively. In the widely used CRISPR/Cas9 system, the endonuclease Cas9 is targeted by a CRISPR small RNA to DNA sequence of interest. In this review, we describe the four available types of genome editing tools, ZFN, TALEN, CRISPR/Cas9 and CRISPR/Cpf1, and show their applications in functional genomics research and precision molecular breeding of crops.     

Mutation in Brca2 stimulates error-prone homology-directed repair of DNA double-strand breaks occurring between repeated sequences

Mutation of BRCA2 causes familial early onset breast and ovarian cancer. BRCA2 has been suggested to be important for the maintenance of genome integrity and to have a role in DNA repair by homology- directed double-strand break (DSB) repair. By studying the repair of a specific induced chromosomal DSB we show that loss of Brca2 leads to a substantial increase in error-prone repair by homology-directed single-strand annealing and a reduction in DSB repair by conservative gene conversion. These data demonstrate that loss of Brca2 causes misrepair of chromosomal DSBs occurring between repeated sequences by stimulating use of an error-prone homologous recombination pathway. Furthermore, loss of Brca2 causes a large increase in genome-wide error-prone repair of both spontaneous DNA damage and mitomycin C-induced DNA cross-links at the expense of error-free repair by sister chromatid recombination. This provides insight into the mechanisms that induce genome instability in tumour cells lacking BRCA2.     

The repair of G-quadruplex-induced DNA damage

G4 DNA motifs, which can form stable secondary structures called G-quadruplexes, are ubiquitous in eukaryotic genomes, and have been shown to cause genomic instability. Specialized helicases that unwind G-quadruplexes in vitro have been identified, and they have been shown to prevent genetic instability in vivo. In the absence of these helicases, G-quadruplexes can persist and cause replication fork stalling and collapse. Translesion synthesis (TLS) and homologous recombination (HR) have been proposed to play a role in the repair of this damage, but recently it was found in the nematode Caenorhabditis elegans that G4-induced genome alterations are generated by an error-prone repair mechanism that is dependent on the A-family polymerase Theta (Pol θ). Current data point towards a scenario where DNA replication blocked at G-quadruplexes causes DNA double strand breaks (DSBs), and where the choice of repair pathway that can act on these breaks dictates the nature of genomic alterations that are observed in various organisms.     

A role for nuclear envelope–bridging complexes in homology-directed repair

Unless efficiently and faithfully repaired, DNA double-strand breaks (DSBs) cause genome instability. We implicate a Schizosaccharomyces pombe nuclear envelope–spanning linker of nucleoskeleton and cytoskeleton (LINC) complex, composed of the Sad1/Unc84 protein Sad1 and Klarsicht/Anc1/SYNE1 homology protein Kms1, in the repair of DSBs. An induced DSB associates with Sad1 and Kms1 in S/G2 phases of the cell cycle, connecting the DSB to cytoplasmic microtubules. DSB resection to generate single-stranded DNA and the ATR kinase drive the formation of Sad1 foci in response to DNA damage. Depolymerization of microtubules or loss of Kms1 leads to an increase in the number and size of DSB-induced Sad1 foci. Further, Kms1 and the cytoplasmic microtubule regulator Mto1 promote the repair of an induced DSB by gene conversion, a type of homology-directed repair. kms1 genetically interacts with a number of genes involved in homology-directed repair; these same gene products appear to attenuate the formation or promote resolution of DSB-induced Sad1 foci. We suggest that the connection of DSBs with the cytoskeleton through the LINC complex may serve as an input to repair mechanism choice and efficiency.     

Blunt-ended DNA double-strand breaks induced by endonucleases PvuII and EcoRV are poor substrates for repair in Saccharomyces cerevisiae

Most mechanistic studies of repair of DNA double-strand breaks (DSBs) produced by in vivo expression of endonucleases have utilized enzymes that produce cohesive-ended DSBs such as HO, I-SceI and EcoRI. We have developed systems for expression of PvuII and EcoRV, nucleases that produce DSBs containing blunt ends, using a modified GAL1 promoter that has reduced basal activity. Expression of PvuII and EcoRV caused growth inhibition and strong cell killing in both haploid and diploid yeast cells. Surprisingly, there was little difference in sensitivities of wildtype cells and mutants defective in homologous recombination, nonhomologous end-joining (NHEJ), or both pathways. Physical analysis using standard and pulsed field gel electrophoresis demonstrated time-dependent breakage of chromosomal DNA within cells. Although ionizing radiation-induced DSBs were largely repaired within 4 h, no repair of PvuII-induced breaks could be detected in diploid cells, even after arrest in G2/M. Rare survivors of PvuII expression had an increased frequency of chromosome XII deletions, an indication that a fraction of the induced DSBs could be repaired by an error-prone process. These results indicate that, unlike DSBs with complementary single-stranded DNA overhangs, blunt-ended DSBs in yeast chromosomes are poor substrates for repair by either NHEJ or recombination.     

Longevity and resistance to stress correlate with DNA repair capacity in Caenorhabditis elegans

DNA repair is an important mechanism by which cells maintain genomic integrity. Decline in DNA repair capacity or defects in repair factors are thought to contribute to premature aging in mammals. The nematode Caenorhabditis elegans is a good model for studying longevity and DNA repair because of key advances in understanding the genetics of aging in this organism. Long-lived C. elegans mutants have been identified and shown to be resistant to oxidizing agents and UV irradiation, suggesting a genetically determined correlation between DNA repair capacity and life span. In this report, gene-specific DNA repair is compared in wild-type C. elegans and stress-resistant C. elegans mutants for the first time. DNA repair capacity is higher in long-lived C. elegans mutants than in wild-type animals. In addition, RNAi knockdown of the nucleotide excision repair gene xpa-1 increased sensitivity to UV and reduced the life span of long-lived C. elegans mutants. These findings support that DNA repair capacity correlates with longevity in C. elegans.     

Efficient disruption of endogenous Bombyx gene by TAL effector nucleases

Engineered nucleases are proteins that are able to cleave DNA at specified sites in the genome. These proteins have recently been used for gene targeting in a number of organisms. We showed earlier that zinc finger nucleases (ZFNs) can be used for generating gene-specific mutations in Bombyx mori by an error-prone DNA repair process of non-homologous end joining (NHEJ). Here we test the utility of another type of chimeric nuclease based on bacterial TAL effector proteins in order to induce targeted mutations in silkworm DNA. We designed three TAL effector nucleases (TALENs) against the genomic locus BmBLOS2, previously targeted by ZFNs. All three TALENs were able to induce mutations in silkworm germline cells suggesting a higher success rate of this type of chimeric enzyme. The efficiency of two of the tested TALENs was slightly higher than of the successful ZFN used previously. Simple design, high frequency of candidate targeting sites and comparable efficiency of induction of NHEJ mutations make TALENs an important alternative to ZFNs.     

DNA double-strand break repair in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Faithful repair of DNA double-strand breaks (DSBs) is vital for animal development, as inappropriate repair can cause gross chromosomal alterations that result in cellular dysfunction, ultimately leading to cancer, or cell death. Correct processing of DSBs is not only essential for maintaining genomic integrity, but is also required in developmental programs, such as gametogenesis, in which DSBs are deliberately generated. Accordingly, DSB repair deficiencies are associated with various developmental disorders including cancer predisposition and infertility. To avoid this threat, cells are equipped with an elaborate and evolutionarily well-conserved network of DSB repair pathways. In recent years, Caenorhabditis elegans has become a successful model system in which to study DSB repair, leading to important insights in this process during animal development. This review will discuss the major contributions and recent progress in the C. elegans field to elucidate the complex networks involved in DSB repair, the impact of which extends well beyond the nematode phylum.     

Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease

Homologous recombination (HR) is a highly accurate mechanism of DNA repair that can be exploited for homology-directed gene targeting. Since in most cell types HR occurs very infrequently (∼10⁻⁶ to 10⁻⁸), its practical application has been largely restricted to specific experimental systems that allow selection of the few cells that become genetically modified. HR-mediated gene targeting has nonetheless revolutionized genetics by greatly facilitating the analysis of mammalian gene function. Recent studies showed that generation of double-strand DNA breaks at specific loci by designed endonucleases greatly increases the rate of homology-directed gene repair. These findings opened new perspectives for HR-based genome editing in higher eukaryotes. Here, we demonstrate by using donor DNA templates together with the adeno-associated virus (AAV) Rep78 and Rep68 proteins that sequence- and strand-specific cleavage at a native, predefined, human locus can also greatly enhance homology-directed gene targeting. Our findings argue for the development of other strategies besides direct induction of double-strand chromosomal breaks to achieve efficient and heritable targeted genetic modification of cells and organisms. Finally, harnessing the cellular HR pathway through Rep-mediated nicking expands the range of strategies that make use of AAV elements to bring about stable genetic modification of human cells.     

Cas9-assisted recombineering in C. elegans: genome editing using in vivo assembly of linear DNAs

Recombineering, the use of endogenous homologous recombination systems to recombine DNA in vivo, is a commonly used technique for genome editing in microbes. Recombineering has not yet been developed for animals, where non-homology-based mechanisms have been thought to dominate DNA repair. Here, we demonstrate, using Caenorhabditis elegans, that linear DNAs with short homologies (∼35 bases) engage in a highly efficient gene conversion mechanism. Linear DNA repair templates with homology to only one side of a double-strand break (DSB) initiate repair efficiently, and short overlaps between templates support template switching. We demonstrate the use of single-stranded, bridging oligonucleotides (ssODNs) to target PCR fragments for repair of DSBs induced by CRISPR/Cas9 on chromosomes. Based on these findings, we develop recombineering strategies for precise genome editing that expand the utility of ssODNs and eliminate in vitro cloning steps for template construction. We apply these methods to the generation of GFP knock-in alleles and gene replacements without co-integrated markers. We conclude that, like microbes, metazoans possess robust homology-dependent repair mechanisms that can be harnessed for recombineering and genome editing.     

Early steps of double-strand break repair in Bacillus subtilis

All organisms rely on integrated networks to repair DNA double-strand breaks (DSBs) in order to preserve the integrity of the genetic information, to re-establish replication, and to ensure proper chromosomal segregation. Genetic, cytological, biochemical and structural approaches have been used to analyze how Bacillus subtilis senses DNA damage and responds to DSBs. RecN, which is among the first responders to DNA DSBs, promotes the ordered recruitment of repair proteins to the site of a lesion. Cells have evolved different mechanisms for efficient end processing to create a 3′-tailed duplex DNA, the substrate for RecA binding, in the repair of one- and two-ended DSBs. Strand continuity is re-established via homologous recombination (HR), utilizing an intact homologous DNA molecule as a template. In the absence of transient diploidy or of HR, however, two-ended DSBs can be directly re-ligated via error-prone non-homologous end-joining. Here we review recent findings that shed light on the early stages of DSB repair in Firmicutes.     

Genome-editing technologies for gene correction of hemophilia

Hemophilia is caused by various mutations in blood coagulation factor genes, including factor VIII (FVIII) and factor IX (FIX), that encode key proteins in the blood clotting pathway. Although the addition of therapeutic genes or infusion of clotting factors may be used to remedy hemophilia’s symptoms, no permanent cure for the disease exists. Moreover, patients often develop neutralizing antibodies or experience adverse effects that limit the therapy’s benefits. However, targeted gene therapy involving the precise correction of these mutated genes at the genome level using programmable nucleases is a promising strategy. These nucleases can induce double-strand breaks (DSBs) on genomes, and repairs of such induced DSBs by the two cellular repair systems enable a targeted gene correction. Going beyond cultured cell systems, we are now entering the age of direct gene correction in vivo using various delivery tools. Here, we describe the current status of in vivo and ex vivo genome-editing technology related to potential hemophilia gene correction and the prominent issues surrounding its application in patients with monogenic diseases.     

Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects

Engineered zinc finger nucleases (ZFNs) induce DNA double-strand breaks at specific recognition sequences and can promote efficient introduction of desired insertions, deletions or substitutions at or near the cut site via homology-directed repair (HDR) with a double- and/or single-stranded donor DNA template. However, mutagenic events caused by error-prone non-homologous end-joining (NHEJ)-mediated repair are introduced with equal or higher frequency at the nuclease cleavage site. Furthermore, unintended mutations can also result from NHEJ-mediated repair of off-target nuclease cleavage sites. Here, we describe a simple and general method for converting engineered ZFNs into zinc finger nickases (ZFNickases) by inactivating the catalytic activity of one monomer in a ZFN dimer. ZFNickases show robust strand-specific nicking activity in vitro. In addition, we demonstrate that ZFNickases can stimulate HDR at their nicking site in human cells, albeit at a lower frequency than by the ZFNs from which they were derived. Finally, we find that ZFNickases appear to induce greatly reduced levels of mutagenic NHEJ at their target nicking site. ZFNickases thus provide a promising means for inducing HDR-mediated gene modifications while reducing unwanted mutagenesis caused by error-prone NHEJ.     

A robust CRISPR–Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break repair

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR–Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.     

Caenorhabditis elegans HIM-18/SLX-4 Interacts with SLX-1 and XPF-1 and Maintains Genomic Integrity in the Germline by Processing Recombination Intermediates

Homologous recombination (HR) is essential for the repair of blocked or collapsed replication forks and for the production of crossovers between homologs that promote accurate meiotic chromosome segregation. Here, we identify HIM-18, an ortholog of MUS312/Slx4, as a critical player required in vivo for processing late HR intermediates in Caenorhabditis elegans. DNA damage sensitivity and an accumulation of HR intermediates (RAD-51 foci) during premeiotic entry suggest that HIM-18 is required for HR–mediated repair at stalled replication forks. A reduction in crossover recombination frequencies—accompanied by an increase in HR intermediates during meiosis, germ cell apoptosis, unstable bivalent attachments, and subsequent chromosome nondisjunction—support a role for HIM-18 in converting HR intermediates into crossover products. Such a role is suggested by physical interaction of HIM-18 with the nucleases SLX-1 and XPF-1 and by the synthetic lethality of him-18 with him-6, the C. elegans BLM homolog. We propose that HIM-18 facilitates processing of HR intermediates resulting from replication fork collapse and programmed meiotic DSBs in the C. elegans germline.