Homologous Recombination In Cells Research Articles

Increased On-Target Rate and Risk of Concatemerization after CRISPR-Enhanced Targeting in ES Cells.

The French mouse clinic (Institut Clinique de la Souris; ICS) has produced more than 2000 targeting vectors for 'à la carte' mutagenesis in C57BL/6N mice. Although most of the vectors were used successfully for homologous recombination in murine embryonic stem cells (ESCs), a few have failed to target a specific locus after several attempts. We show here that co-electroporation of a CRISPR plasmid with the same targeting construct as the one that failed previously allows the systematic achievement of positive clones. A careful validation of these clones is, however, necessary as a significant number of clones (but not all) show a concatemerization of the targeting plasmid at the locus. A detailed Southern blot analysis permitted characterization of the nature of these events as standard long-range 5' and 3' PCRs were not able to distinguish between correct and incorrect alleles. We show that a simple and inexpensive PCR performed prior to ESC amplification allows detection and elimination of those clones with concatemers. Finally, although we only tested murine ESCs, our results highlight the risk of mis-validation of any genetically modified cell line (such as established lines, induced pluripotent stem cells or those used for ex vivo gene therapy) that combines the use of CRISPR/Cas9 and a circular double-stranded donor. We strongly advise the CRISPR community to perform a Southern blot with internal probes when using CRISPR to enhance homologous recombination in any cell type, including fertilized oocytes.

Experience with olaparib in a patient with luminal HER2-positive metastatic breast cancer

Hereditary breast cancer (BC) accounts for about 5-10% of cases. BRCA-associated tumors have been identified as a separate group of malignant neoplasms with distinctive clinical manifestations and specific treatment features. Understanding of biological mechanisms leading to cancer in BRCA1/2 mutation carriers and discovery of potential molecular targets, such as poly (ADP-ribose) polymerase (PARP), involved in base excision repair mechanisms, led to the development of a new class of targeted drugs belonging to the PARP inhibitors group. PARP inhibition leads to the preservation of single-stranded DNA breaks, the arrest of the replication fork, and the realization of the “synthetic lethality” phenomenon due to the inability to repair double-stranded DNA breaks by homologous recombination in cells with mutations in the BRCA1/2 genes. Two randomized trials OlympiAD and EMBRACA evaluated and proved the effectiveness of PARP inhibitors in patients with metastatic BRCA-mutated HER2-negative breast cancer in comparison with standard chemotherapy. At the same time, data on the potential use of PARP inhibitors for the treatment of BRCA-mutated HER2-positive breast cancer patients are extremely limited. This article presents a clinical example of the use of olaparib in a patient with BRCA-mutated HER2-positive metastatic breast cancer.

Irc20 Regulates the Yeast Endogenous 2-μm Plasmid Levels by Controlling Flp1.

The endogenous yeast 2-μm plasmid while innocuous to the host, needs to be properly regulated to avoid a toxic increase in copy number. The plasmid copy number control system is under the control of the plasmid encoded recombinase, Flp1. In case of a drop in 2-μm plasmid levels due to rare plasmid mis-segregation events, the Flp1 recombinase together with the cell’s homologous recombination machinery, produce multiple copies of the 2-μm plasmid that are spooled during DNA replication. The 2-μm plasmid copy number is tightly regulated by controlled expression of Flp1 as well as its ubiquitin and SUMO modification. Here, we identify a novel regulator of the 2-μm plasmid, the ATPase, ubiquitin ligase, Irc20. Irc20 was initially identified as a homologous recombination regulator, and here we uncover a new role for Irc20 in maintaining the 2-μm plasmid copy number and segregation through regulating Flp1 protein levels in the cell.

Simultaneous Delivery of CRISPR/Cas and Donor DNA using Cell‐Penetrating Peptide‐Adaptors

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are utilized as a form of adaptive immunity in prokaryotes. CRISPR detects and eliminates foreign nucleic acids such as those found in bacteriophage. To achieve this form of ‘memory of infection,’ CRISPR associated proteins (Cas) form complexes with RNA to cause double-stranded breaks (DSBs) in the invader's sequence. The invader's DNA between these breaks is incorporated within the CRISPR locus in the bacterial genome. CRISPR/Cas9 has been a breakthrough for genome editing due to its specificity and potential to allow genome editing in patients, where it could be used to treat many diseases such as cancer or hemophilia. The present work aims to enhance CRISPR efficiency by delivery of Cas9 complexed with guide RNA (crRNA and tracRNA) to the nuclei of living cells using a novel cell-penetrating peptide (CPP)-adaptor system. CPPs have long been used to deliver biomolecules to living cells, but CPP-cargos remained trapped in endosomes. To solve this problem, we used a novel CPP adaptor, TAT-CaM, a fusion protein in which the CPP from HIV TAT is fused to human calmodulin (CaM). When Ca2+ is present, TAT-CaM binds to an engineered calmodulin binding site (CBS) on cargo proteins (e.g. CBS-Cas9). After endocytosis, TAT-CaM dissociates from cargos as Ca2+ concentrations drop during trafficking. Like other CPPs, TAT-CaM remains trapped in endosomes but cargos efficiently escape. In the present study, biolayer interferometry (BLI) and confocal microscopy were used to demonstrate Ca2+-dependent binding and delivery of CRISPR complexes to the nuclei of cells. We have employed a similar scheme to deliver in parallel donor DNA that can be used by the cell's homologous recombination machinery to effect editing. Donor DNA is internalized simultaneously with CRISPR/Cas using a CBS-tamavidin fusion protein that binds both TAT-CaM and biotinylated donor DNA. Preliminary results show that CBS-tamavidin is wellexpressed in E. coli and readily purified using immobilized metal affinity chromatography. BLI analysis showed that CBS-tamavidin binds TAT-CaM affinity in the presence of Ca2+ while simultaneously binding biotinylated DNA, both with high affinity (fast on/slow off). Confocal microscopy showed that CBS-Cas9 and CBS-tamavidin/biotin-DNA enter cells in the presence TAT-CaM but not in the absence. Support or Funding Information National Institutes of Health grant GM R15120691 Preliminary results for delivery of CBS-Cas9 in Baby Hamster Kidney(BHK) cells using confocal microscopy. CRISPR RNAs are labeled with Alexa 550 red, the nucleus is labeled with NucBlue, the Cas9 and TAT CaM are unlabeled. The first column only shows the CRISPR RNA, the second column only shows the nucleus and the third column shows the composite of the first and the second column. The first row and the second row show the cells with CRISPR complex in 10 and 20 minutes respectively. BioLayer Interferometry (BLI) analysis of CBS-Cas9. (A) Association and dissociation of CBS-Cas9 (analyte at concentrations starting from 200nM to 50nM) in TAT CaM(ligand at 1uM) and a calcium-containing buffer respectively. (B) CBS-Cas9 dissociation in EDTA(chelates calcium) from 200nM to 50nM. (C) Kinetic parameters for CBS-Cas9 from A and B. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

To Nick or Not to Nick: Comparison of I-SceI Single- and Double-Strand Break-Induced Recombination in Yeast and Human Cells

Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, or chromosomal rearrangements. By contrast, generation of a DNA single-strand break (SSB), or nick, can stimulate gene correction without the problems of DSB repair because the uncut DNA strand acts as a template to permit healing without alteration of genetic material. Here, we examine the ability of a nicking variant of the I-SceI endonuclease (K223I I-SceI) to stimulate gene targeting in yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK-293) cells. K223I I-SceI is proficient in both yeast and human cells and promotes gene correction up to 12-fold. We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick. We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells. These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy.

TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain

DNA double-strand breaks enhance homologous recombination in cells and have been exploited for targeted genome editing through use of engineered endonucleases. Here we report the creation and initial characterization of a group of rare-cutting, site-specific DNA nucleases produced by fusion of the restriction enzyme FokI endonuclease domain (FN) with the high-specificity DNA-binding domains of AvrXa7 and PthXo1. AvrXa7 and PthXo1 are members of the transcription activator-like (TAL) effector family whose central repeat units dictate target DNA recognition and can be modularly constructed to create novel DNA specificity. The hybrid FN-AvrXa7, AvrXa7-FN and PthXo1-FN proteins retain both recognition specificity for their target DNA (a 26 bp sequence for AvrXa7 and 24 bp for PthXo1) and the double-stranded DNA cleaving activity of FokI and, thus, are called TAL nucleases (TALNs). With all three TALNs, DNA is cleaved adjacent to the TAL-binding site under optimal conditions in vitro. When expressed in yeast, the TALNs promote DNA homologous recombination of a LacZ gene containing paired AvrXa7 or asymmetric AvrXa7/PthXo1 target sequences. Our results demonstrate the feasibility of creating a tool box of novel TALNs with potential for targeted genome modification in organisms lacking facile mechanisms for targeted gene knockout and homologous recombination.

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

The sequencing of the human genome has greatly facilitated our ability to identify the gene candidates that are critical for developmental regulation and disease progression. However, investigators seeking to use such data to gain a better understanding of the developmental biology and therapeutic potential of pluripotent stem cell types may be frustrated by the limitations of current genetic engineering techniques. Targeted genomic manipulation of mammalian cells is inefficient, and unintended effects of such manipulation on the cells’ developmental and proliferative potential remain uninvestigated. To this end, the recent demonstration of targeted gene correction in human induced pluripotent stem cells (hiPSCs) before and after reprogramming, as reported in this issue of Molecular Therapy, warrants special mention.1

Contributions of Nucleotide Excision Repair, DNA Polymerase η, and Homologous Recombination to Replication of UV-irradiated Herpes Simplex Virus Type 1

The effects of UV irradiation on herpes simplex virus type 1 (HSV-1) gene expression and DNA replication were examined in cell lines containing mutations inactivating the XPA gene product required for nucleotide-excision repair, the DNA polymerase eta responsible for translesion synthesis, or the Cockayne syndrome A and B (CSA and CSB) gene products required for transcription-coupled nucleotide excision repair. In the absence of XPA and CSA and CSB gene products, virus replication was reduced 10(6)-, 400-, and 100-fold, respectively. In DNA polymerase eta mutant cells HSV-1 plaque efficiency was reduced 10(4)-fold. Furthermore, DNA polymerase eta was strictly required for virus replication at low multiplicities of infection but dispensable at high multiplicities of infection. Knock down of Rad 51, Rad 52, and Rad 54 levels by RNA interference reduced replication of UV-irradiated HSV-1 150-, 100-, and 50-fold, respectively. We find that transcription-coupled repair efficiently supports expression of immediate early and early genes from UV-irradiated HSV-1 DNA. In contrast, the progression of the replication fork appears to be impaired, causing a severe reduction of late gene expression. Since the HSV-1 replisome does not make use of proliferating cell nuclear antigen, we attribute the replication defect to an inability to perform proliferating cell nuclear antigen-dependent translesion synthesis by polymerase switching at the fork. Instead, DNA polymerase eta may act during postreplication gap filling. Homologous recombination, finally, might restore the physical and genetic integrity of the virus chromosome.

Effects of liposome-mediated rat cyclooxygenase-2 antisense RNA on growth of hepatocarcinoma cells

Objective To construct a recombinant eukaryotic expression vector encoding rat COX-2 antisense RNA and investigate its effects on rat liver cancer cell proliferatiion. Methods The plasmid encoding anti-sense COX-2 was constructed by using cloning COX-2 cDNA fragment in the reverse direction into the pcDNA3. 1. Then the plasmid pcDNA3. l/COX-2as was transfered into rat hepatocacinoma cell line CBRH7919 with liposome and homologous recombination cell was named as CBRH7919-A. The cell proliferation, cell cycle and apoptosis were analyzed by MTT, flow cytometry and RT-PCR. CBRH7919 cells with or without pretreatment were inoculated into nude mice and the cell proliferation was observed in vivo. Results CBRH7919 treated with antisense COX-2 greatly inhibited the proliferation with a rate of 78% and DNA synthesis (PI of COX-2 group vs control group, 0. 361 vs 0. 784) of CBRH7919 cell line, decreased coloning formation in anchorage-independent assay. In vivo tumorigenic rate was greatly reduced in athymic nude mice as compared with untreated cell group (25% vs 100%). Apoptosis-related gene expression was not different as compared with untreated cell group (P>0. 05). Conclusion Antisense COX-2 RNA can inhibit the proliferation of rat liver cancer cell line in vitro as well as in vivo, suggesting that it has potential value in hepatocellular cancer gene therapy. Key words: Carcinoma hepatocellular; Liposome; Rat; Cyclooxygenase-2; Antisense RNA

Homing endonucleases: from basics to therapeutic applications.

Homing endonucleases (HE) are double-stranded DNAses that target large recognition sites (12-40 bp). HE-encoding sequences are usually embedded in either introns or inteins. Their recognition sites are extremely rare, with none or only a few of these sites present in a mammalian-sized genome. However, these enzymes, unlike standard restriction endonucleases, tolerate some sequence degeneracy within their recognition sequence. Several members of this enzyme family have been used as templates to engineer tools to cleave DNA sequences that differ from their original wild-type targets. These custom HEs can be used to stimulate double-strand break homologous recombination in cells, to induce the repair of defective genes with very low toxicity levels. The use of tailored HEs opens up new possibilities for gene therapy in patients with monogenic diseases that can be treated ex vivo. This review provides an overview of recent advances in this field.

300. Targeted Gene Correction in Mouse Embryonic Stem Cells Using Helper-Dependent Adenoviral Vectors

Integration of a therapeutic gene into random sites on host chromosomes could potentially lead to serious problems such as cellular transformation and gene silencing. Gene targeting, the process of homologous recombination between exogenous and chromosomal DNA, would achieve safe, transcriptionally active and stable expression of an integrated gene. However, the low frequency of homologous recombination in mammalian cells prevents therapeutic application of this strategy. Recombinant adenoviral vectors (AdVs) might be an ideal vehicle for homologous recombination because of their highly efficient delivery of double-stranded DNA genomes into the nucleus of almost any cell type. We examined the ability of AdVs to correct an insertional mutation in exon 3 of the hypoxanthine phosphoribosyl transferase (Hprt) locus in male mouse embryonic stem (ES) cells. The cells were infected with four types of Hprt targeting AdVs (E1-deleted vectors with 1.7-kb and 6.7-kb wild-type Hprt sequences and helper-dependent (HD) AdVs with 6.7-kb and 18.6-kb wild-type Hprt sequences) at multiplicities of infection (MOI) of 10, 100 and 1000 genomes per cell. The ES cells that might have undergone homologous recombination and thus restored their HPRT activity were detected by HAT selection. The HD AdVs also encode the beta-geo marker gene, which flanks the Hprt sequence. Therefore, randomly integrated vectors could also be detected by G418 selection. The frequency of homologous recombination in cells infected with HD AdVs with an 18.6-kb homology was nearly 0.02%, an 800-fold increase compared to those cells infected by an E1-deleted vector with the 6.7-kb homology. No homologous recombination was detected with the E1-deleted vector with the 1.7-kb Hprt sequence. In contrast to the homologous recombination frequency, which showed an MOI-dependent increase, the random integration frequency reached maximum levels at an MOI of 100 and was similar between the HD AdVs. The ratio of random to targeted integration was 61% with the 18.6-kb HD AdV at an MOI of 1000. Treatment of the infected cells with trichostatin A, a histone deacetylase inhibitor, enhanced only the random integration frequency but had no effect on the frequency of targeted integration. The majority of HAT-resistant cell clones showed a pattern of faithful gene targeting. No randomly integrated vectors were observed in the cells with targeted vector integration, as analyzed by PCR. These findings suggest that adenovirus-mediated homologous recombination might be applicable for ex vivo gene therapy.

HIV-1 Tat increases cell survival in response to cisplatin by stimulating Rad51 gene expression.

Tat is an early regulatory protein of human immunodeficiency virus type 1, which plays a central role in the pathogenesis of AIDS by stimulating transcription of the viral genome and impairing several important cellular pathways during the progression of the disease. Here, we investigated the effect of Tat on cell response to DNA damage. Our results indicate that Tat production causes a noticeable increase in the survival rate of PC12 cells upon their treatment with genotoxic agents. Single-cell gel electrophoresis studies revealed reduced DNA breakage in PC12-Tat cells upon cisplatin treatment relative to the control cells. Furthermore, cytogenetic data exhibited less chromosomal damage in Tat-producing cells after recovery from cisplatin treatment, corroborating electrophoretic data. Examination of several proteins involved in the control of DNA repair showed elevated levels of Rad51, a key regulator of homologous recombination in cells expressing Tat. On the other hand, the level of Ku70, one of the components of the nonhomologous end-joining repair pathway, was slightly decreased in cells expressing Tat. Using a fluorescence-based assay, we demonstrated that repair of DNA double-strand breaks via homologous recombination is increased in Tat-producing cells. The results from in vitro nonhomologous end-joining assay revealed a reduced ability of protein extract from PC12-Tat cells compared to PC12 cells in rejoining linearized DNA. These observations ascribe a new role for Tat in host genomic integrity, perhaps by affecting the expression of genes involved in DNA repair.

A new method (GOREC) for directed mutagenesis and gene repair by homologous recombination.

Directed mutagenesis in mammalian cells has been the focus of intense research because of its promising application for gene correction and engineering. Both natural and modified oligonucleotides (ODN), RNA-DNA chimeric oligonucleotide (RDO) and small fragment DNA (SFHR), as well as vector DNA were used for promoting homologous replacement with varying success. It was recently shown that a triple helix-forming oligonucleotide (TFO) tethered to an oligonucleotide (donor DNA) can enhance mutagenesis by homologous recombination in cells. The basic idea is to accelerate homology search by oligonucleotide-directed triple helix formation in the vicinity of the target site for donor DNA. Here we describe a new method named GOREC (guided homologous recombination) which shares similar gene targeting, but has notable difference in the concept with the previous method. It is made of a homing device (TFO) and a donor DNA for effecting distinct functions. They are linked together by non-covalent or covalent interaction. This modular concept allows guidance of either an oligonucleotide (ODN, RDO) or a small DNA fragment to the target site for homologous replacement. Therefore, the triple helix site can be hundreds of base pairs away from the target site. An episomal assay for proof-of-principle study will be presented and discussed.

Domain Analysis of an Archaeal RadA Protein for the Strand Exchange Activity

Archaeal RadA, like eukaryotic Rad51 and bacterial RecA, promotes strand exchange between DNA strands with homologous sequences in vitro and is believed to participate in the homologous recombination in cells. The amino acid sequences of the archaeal RadA proteins are more similar to the eukaryotic Rad51s rather than the bacterial RecAs, and the N-terminal region containing domain I is conserved among the RadA and Rad51 proteins but is absent from RecA. To understand the structure-function relationship of RadA, we divided the RadA protein from Pyrococcus furiosus into two parts, the N-terminal one-third (RadA-n) and the residual C-terminal two-thirds (RadA-c), the latter of which contains the central core domain (domain II) of the RecA/Rad51 family proteins. RadA-c had the DNA-dependent ATPase activity and the strand exchange activity by itself, although much weaker (10%) than that of the intact RadA. These activities of RadA-c were restored to 60% of those of RadA by addition of RadA-n, indicating that the proper active structure of RadA was reconstituted in vitro. These results suggest that the basic activities of the RecA/Rad51 family proteins for homologous recombination are derived from domain II, and the N-terminal region may help to enhance the catalytic efficiencies.

Physical Interaction between Human RAD52 and RPA Is Required for Homologous Recombination in Mammalian Cells

The yeast RAD52 protein is essential for DNA double-strand break repair, and meiotic and mitotic recombination. RPA is a protein complex of three subunits (70, 34, and 11 kDa) that has been shown to be involved in DNA replication, nucleotide excision repair, and homologous recombination. Here, we demonstrate a physical interaction between human RAD52 and RPA in vivo and in vitro. In addition, the domain (amino acids 221-280) in RAD52 protein that mediates the interaction with the 34-kDa subunit of RPA was also determined. Overexpression of mutant RAD52 proteins lacking the interaction domain (amino acids 221-240, 241-260, and 261-280) failed to induce homologous recombination in monkey cells. We have previously shown that overexpression of human RAD52 induced homologous recombination in these cells. These results suggest that direct physical interactions between RAD52 and RPA are essential for homologous recombination in mammalian cells.

Exon organization of the human FKBP-12 gene: correlation with structural and functional protein domains

FKBP-12, the major T-cell binding protein for the immunosuppressive agents FK506 and rapamycin, catalyzes the interconversion of the cis and trans rotamers of the peptidyl-prolyl amide bond of peptide and protein substrates. The function of rotamase activity in cells and the role of FKBP-12 in immunoregulation is uncertain. In this paper we report the cloning and characterization of the human chromosomal FKBP-12 gene and four processed FKBP-12 pseudogenes. The FKBP-12 gene is 24 kilobases in length and contains five exons. The protein-coding region of the gene is divided into four exon modules that correlate with the structural and functional domains of the protein. The novel structure of FKBP-12 resulting from the topology of the antiparallel beta-sheet is the topological crossing of two loops that are encoded by separate exons. Separate exons also encode the antiparallel beta-sheet and alpha-helical region that define the drug-binding pocket and enzyme activity site of FKBP-12. The exon organization of the FKBP-12 gene also provided insight into the genetic evolution of the immunophilin family. Knowledge of the FKBP-12 gene structure will enable inactivation of this gene by homologous recombination in cells to provide a model to study the role of FKBP-12 in immunoregulation and normal cellular processes.

Intramolecular homologous recombination in cells infected with temperature-sensitive mutants of vaccinia virus

I have used a plasmid containing two copies of the Saccharmyces cerevisiae his3 gene to study intramolecular homologous recombination in vaccina virus-infected cells. Recombination of the plasmid was monitored by restriction enzyme digestion and Southern blot hybridization in cells infected with representatives from each of 32 complementation groups of temperature-sensitive mutants ts42 and ts17 did not replicate nor detectably recombine the input plasmid. All except one of the mutants that synthesized normal amounts of viral DNA and protein replicated and recombined the plasmid in a manner indistinguishable from wild-type virus. The remaining mutant, ts13, only poorly replicated and recombined the input plasmid. Thus, the processes of replication and recombination could not be separated by using this battery of mutants. Viral mutants defective in late protein synthesis were unable to resolve the vaccinia virus concatemer junction in plasmids but carried out intramolecular homologous recombination with plasmids as efficiently as did wild-type virus at the conditionally lethal temperature. This result distinguishes homologous recombination, which requires early gene products, from resolution of concatemer junctions, which requires additional late gene products.

Vaccinia virus: a selectable eukaryotic cloning and expression vector.

Foreign DNA was inserted into two nonessential regions of the vaccinia virus genome by homologous recombination in cells infected with virus and transfected with plasmids containing the foreign DNA elements flanked by vaccinia virus DNA. Thymidine kinase-negative (TK-) recombinants were selected after inserting foreign DNA into the coding region of the TK gene of wild-type vaccinia virus; TK+ recombinants were selected after inserting the herpesvirus TK gene into TK- mutants of vaccinia virus. For TK+ expression, it was necessary to insert a 275-base-pair DNA fragment containing the initiation site and sequences upstream of an early vaccinia virus transcript next to the coding sequences of the herpesvirus gene. The unique ability of the herpesvirus TK to phosphorylate 125I-labeled deoxycytidine provided independent confirmation of gene expression. These studies demonstrate the use of vaccinia virus as a selectable cloning and expression vector, confirm the map location of the vaccinia virus TK gene, and provide initial information regarding the location of vaccinia virus transcriptional regulatory sequences.