The precision strategy of human genome correction via a set of circular donor DNA and its cleaver.

Expanding or Restricting the Target Site Repertoire of Zinc-finger Nucleases: The Inter-domain Linker as a Major Determinant of Target Site Selectivity

When DNA is introduced into eukaryotic cells, it can be integrated into the genome by homologous or illegitimate recombination 1,2. Despite great efforts to gain insight into the molecular mechanisms, our understanding of the recombination process is still in its infancy. In the absence of a molecular model, predictions concerning the frequency of homologous recombination compared to illegitimate recombination cannot be precisely made. In mammalian cells, illegitimate recombination is the most predominant event (for review, seeref. (3). Thus, if the integration of DNA via homologous recombination into mammalian cells is the goal of the experiment, a single homologous recombination event has to be detected among many illegitimate recombination events. Described here is a method of detecting homologous recombination events in a small subpopulation of cells by using the polymerase chain reaction (PCR), a primer-directed enzymatic amplification of specific DNA sequences. This method can be used to determine the homologous recombination frequency and to facilitate the cloning of homologously recombined cells 1,2. Homologously recombined alleles are identified by their amplification products, which are generated by the PCR reaction (see Fig. 1). The specificity of the reaction is dependen t on the two primers. One primer (primer 2 in Fig. 1) primes DNA synthesis specifically at the nonhomologous sequences of the exogenous DNA. The other primer (primer 1 in Fig. 1) is specific for the target locus, but outside of the exogenous DNA. Thus, both priming sites are physically linked in a predictable manner only after homologous recombination. Fig. 1. Detection of homologous recombination events by PCR. Homologous recombination between a target locus and exogenous DNA yields a new recombinant molecule in which a small portion of the target locus is replaced by a heterologous sequence of the exogenous DNA. This recombination event links two priming sites for the PCR. Primer 1 primes DNA synthesis from the target sequence, outside of the region homologous to the exogenous DNA. Primer 2 is specific for the heterologous sequence residing in the exogenous DNA. These priming sites are not linked (in the same manner) after the integration of the exogenous DNA by illegitimate recombination. Yet, DNAs of a heterogeneous length are made from the target and the illegitimately recombined sequence during every PCR cycle. Those molecules that terminate in the region of homology could anneal, thus creating a recombinant molecule.

Coupling homologous recombination with growth selection in yeast: a tool for construction of random DNA sequence libraries.

CRISPR/Cas9, which generates DNA double-strand breaks (DSBs) at target loci, is a powerful tool for editing genomes when codelivered with a donor DNA template. However, DSBs, which are the most deleterious type of DNA damage, often result in unintended nucleotide insertions/deletions (indels) via mutagenic nonhomologous end joining. We developed a strategy for precise gene editing that does not generate DSBs. We show that a combination of single nicks in the target gene and donor plasmid (SNGD) using Cas9D10A nickase promotes efficient nucleotide substitution by gene editing. Nicking the target gene alone did not facilitate efficient gene editing. However, an additional nick in the donor plasmid backbone markedly improved the gene-editing efficiency. SNGD-mediated gene editing led to a markedly lower indel frequency than that by the DSB-mediated approach. We also show that SNGD promotes gene editing at endogenous loci in human cells. Mechanistically, SNGD-mediated gene editing requires long-sequence homology between the target gene and repair template, but does not require CtIP, RAD51, or RAD52. Thus, it is considered that noncanonical homology-directed repair regulates the SNGD-mediated gene editing. In summary, SNGD promotes precise and efficient gene editing and may be a promising strategy for the development of a novel gene therapy approach.

Following V(D)J cleavage, the newly liberated DNA signal ends can be either fused together into a signal joint or used as donor DNA in RAG-mediated transposition. We find that both V(D)J cleavage and release of flanking coding DNA occur before the target capture step of transposition can proceed; no coding DNA is ever detected in the target capture complex. Separately from its role in V(D)J cleavage, the DDE motif of the RAG1/2 active site is specifically required for target DNA capture. The requirement for cleavage and release of coding DNA prior to either physical target binding or functional target commitment suggests that the RAG1/2 transposase contains a single binding site for non-RSS DNA that can accommodate either target DNA or coding DNA, but not both together. Perhaps the presence of coding DNA may aid in preventing transpositional resolution of V(D)J recombination intermediates.

The intranuclear location of genomic loci and the dynamics of these loci are important parameters for understanding the spatial and temporal regulation of gene expression. Recently it has proven possible to visualize endogenous genomic loci in live cells by the use of transcription activator-like effectors (TALEs), as well as modified versions of the bacterial immunity clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Here we report the design of multicolor versions of CRISPR using catalytically inactive Cas9 endonuclease (dCas9) from three bacterial orthologs. Each pair of dCas9-fluorescent proteins and cognate single-guide RNAs (sgRNAs) efficiently labeled several target loci in live human cells. Using pairs of differently colored dCas9-sgRNAs, it was possible to determine the intranuclear distance between loci on different chromosomes. In addition, the fluorescence spatial resolution between two loci on the same chromosome could be determined and related to the linear distance between them on the chromosome's physical map, thereby permitting assessment of the DNA compaction of such regions in a live cell.

To determine the minimal DNA sequence homology required for recombination in Bacillus subtilis, we developed a system capable of distinguishing between homologous and illegitimate recombination events during plasmid integration into the chromosome. In this system the recombination frequencies were measured between ts pE194 derivatives carrying segments of the chromosomal beta-gluconase gene (bglS) of various lengths and the bacterial chromosome, using selection for erythromycin resistance at the non-permissive temperature. Homologous recombination events, resulting in disruption of the bglS gene, were easily detected by a colorimetric assay for beta-gluconase activity. A linear dependence of recombination frequency on homology length was observed over an interval of 77 bp. It was found that approximately 70 bp of homology is required for detectable homologous recombination. Homologous recombination was not detected when only 25 bp of homology between plasmid and chromosome were provided. The data indicate that homology requirements for recombination in B. subtilis differ from those in Escherichia coli.

Insertion sequence (IS) elements are the simplest autonomous transposable elements found in prokaryotic genomes1. We recently discovered that IS110 family elements encode a recombinase and a non-coding bridge RNA (bRNA) that confers modular specificity for target DNA and donor DNA through two programmable loops2. Here we report the cryo-electron microscopy structures of the IS110 recombinase in complex with its bRNA, target DNA and donor DNA in three different stages of the recombination reaction cycle. The IS110 synaptic complex comprises two recombinase dimers, one of which houses the target-binding loop of the bRNA and binds to target DNA, whereas the other coordinates the bRNA donor-binding loop and donor DNA. We uncovered the formation of a composite RuvC–Tnp active site that spans the two dimers, positioning the catalytic serine residues adjacent to the recombination sites in both target and donor DNA. A comparison of the three structures revealed that (1) the top strands of target and donor DNA are cleaved at the composite active sites to form covalent 5′-phosphoserine intermediates, (2) the cleaved DNA strands are exchanged and religated to create a Holliday junction intermediate, and (3) this intermediate is subsequently resolved by cleavage of the bottom strands. Overall, this study reveals the mechanism by which a bispecific RNA confers target and donor DNA specificity to IS110 recombinases for programmable DNA recombination.

The biochemistry of retroviral integration selectivity is not fully understood. We modified the previously reported in vitro integration reaction protocol and developed a novel reaction system with higher efficiency. We used a DNA target composed of a repeat sequence DNA, 5’-(GTCCCTTCCCAGT)6(ACTGGGAAGGGAC) 6-3’, that was ligated into a circular plasmid. Target DNA was reacted with a pre-integration (PI) complex that was formed by incubation of the end cDNA of the HIV-1 genome and recombinant integrase. It was confirmed that integration selectively occurred in the middle segment of the repeat sequence. On the other hand, both frequency and selectivity of integration markedly decreased when target sequences were used in which CAGT bases in the middle position of the original target sequence were deleted. Moreover, upon incubation with a combination of these deleted DNAs and the original sequence, the integration efficiency and selectivity towards the original target sequence were significantly reduced, which indicated interference effects by the deleted sequence DNAs. Efficiency and selectivity were also found to vary with changes in the manganese dichloride concentration of the reaction buffer, probably due to induction of fluctuation in the secondary structure of the substrate DNA. Such fluctuation may generate structural isomers that are favorable for selective integration into the target sequence DNA. In conclusion, there is considerable selectivity in HIV-integration into the specified target sequence. The present in vitro integration system will therefore be useful for monitoring viral integration activity or for testing of integrase inhibitors.

Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination.

The EHA Research Roadmap: Hematopoietic Stem Cell Gene Therapy.

To characterize homologous recombination of transforming DNA in the filamentous fungus Alternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. The A. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus. BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internal BRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Mel-) transformants accounted for 30 to 80% of hygromycin B-resistant (HyR) transformants, correlating closely with the size of the BRM1 segment in the transforming DNA. Restriction enzyme digestion within the BRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of the BRM1 segments. Plasmids carrying both BRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Mel- transformants accounted for only 5% of HyR transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within the BRM1 segment was used, almost all transformants were Mel-. These results indicate that homologous integration of circular molecules in A. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.

Gene Correction in Human Embryonic and Induced Pluripotent Stem Cells: Promises and Challenges Ahead

Genomic rearrangements, encompassing mutational changes in the genome such as insertions, deletions or inversions, are essential for genetic diversity. These rearrangements are typically orchestrated by enzymes that are involved in fundamental DNA repair processes, such as homologous recombination, or in the transposition of foreign genetic material by viruses and mobile genetic elements1,2. Here we report that IS110 insertion sequences, a family of minimal and autonomous mobile genetic elements, express a structured non-coding RNA that binds specifically to their encoded recombinase. This bridge RNA contains two internal loops encoding nucleotide stretches that base-pair with the target DNA and the donor DNA, which is the IS110 element itself. We demonstrate that the target-binding and donor-binding loops can be independently reprogrammed to direct sequence-specific recombination between two DNA molecules. This modularity enables the insertion of DNA into genomic target sites, as well as programmable DNA excision and inversion. The IS110 bridge recombination system expands the diversity of nucleic-acid-guided systems beyond CRISPR and RNA interference, offering a unified mechanism for the three fundamental DNA rearrangements—insertion, excision and inversion—that are required for genome design.

Generating knockout mice is still an expensive and highly time-consuming process. Target construct generation, the first labor-intensive step in this process, requires the manipulation of large fragments of DNA and numerous, and often cumbersome, cloning steps. Here we show the development of a rapid approach for generating targeting constructs that capitalizes on efficient homologous recombination between linear DNA fragments and circular plasmids in Escherichia coli ("recombineering"), the availability of bacterial artificial chromosomes (BACs), and the accessibility of the sequence of the mouse genome. Employing recombineering, we demonstrate with only 1-2 template plasmids, short homologies (40-50bp) between donor and target DNA, and one subcloning step that we can efficiently manipulate BACs in situ to generate a complicated targeting vector. This procedure avoids the need to construct or screen genomic libraries and permits the generation of most standard, conditional, or knock-in targeting vectors, often within two weeks.