Zinc Finger Endonucleases Research Articles

A review on principles, applications and the current challenges to genome editing in insects

Genome editing is an important tool in synthetic biology and the past few decades has witnessed a tremendous pool of know-hows into the intricacies of the process. This technique can introduce altered traits in the organism’s genome that can be extremely beneficial in controlling the spread of dangerous vector-borne diseases that torment our society today like dengue, malaria, chikungunya etc. In the past, random chemical and radiation mutagenesis were performed but now researchers rely on more site-specific mutations using CRISPR Cas, Zinc Finger endonucleases, TALENS, etc. Using gene drives for controlling the spread of the desired trait among the wild population and tackling public health issues are also discussed. The ultimate goal of this review paper is to highlight the major developments that have taken place in the field of genome editing in insects, to put into light the progress and challenges that have been overcome. Ultimately the current limitations and the future possibilities have also been pointed out.

Applications of CRISPR/Cas gene-editing technology in yeast and fungi.

Genome editing technology has progressed rapidly in recent years. Although traditional gene-editing methods, including homologous recombination, zinc finger endonucleases, and transcription activator-like effector nucleases, have substantial implications for research in genetics and molecular biology, but they have remarkable limitations, including their low efficiency, high error rate, and complex design. A new gene-editing technology, the CRISPR/Cas system, was developed based on studies of archaeal and bacterial immune responses to viruses. Owing to its high target efficiency, simple primer design, and wide applications, the CRISPR/Cas system, whose developers were awarded the Nobel Prize in Chemistry in 2020, has become the dominant genomic editing technology in academia and the pharmaceutical industry. Here, we briefly introduce the CRISPR/Cas system and its main applications for genome engineering, metabolic engineering, and transcriptional regulation in yeast, filamentous fungi, and macrofungi. The polygene and polyploid editing, construction of yeast chromosomes, yeast library creation, regulation of metabolic pathways, and CRISPR activation/CRISPR interference systems are mainly summarized and discussed. The potential applications for the treatment of fungal infections and the further transformation and application of the CRISPR/Cas system in fungi are also proposed and discussed.

Ku70 suppresses alternative end joining in G1-arrested progenitor B cells

Classical nonhomologous end joining (C-NHEJ) repairs DNA double-strand breaks (DSBs) throughout interphase but predominates in G1 phase when homologous recombination is unavailable. Complexes containing the Ku70/80 ("Ku") and XRCC4/ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While XRCC4/Lig4 are absolutely required for joining RAG1/2 endonuclease ("RAG")-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Lig4 also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Lig4-mediated joining has been attributed to RAG shepherding V(D)J DSBs exclusively into the C-NHEJ pathway. Here, we report that A-EJ of DSB ends generated by RAG1/2, Cas9:gRNA, and Zinc finger endonucleases in Lig4-deficient G1-arrested progenitor B cell lines is suppressed by Ku. Thus, while diverse DSBs remain largely as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 increases DSB rejoining and translocation levels to those observed in Ku70-deficient counterparts. Correspondingly, while RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient G1-arrested progenitor B cell lines, joining of RAG-generated DSBs in Ku70-deficient and Ku70/Lig4 double-deficient lines occurs through a translocation-like A-EJ mechanism. Thus, in G1-arrested, Lig4-deficient progenitor B cells are functionally end-joining suppressed due to Ku-dependent blockage of A-EJ, potentially in association with G1-phase down-regulation of Lig1. Finally, we suggest that differential impacts of Ku deficiency versus Lig4 deficiency on V(D)J recombination, neuronal apoptosis, and embryonic development results from Ku-mediated inhibition of A-EJ in the G1 cell cycle phase in Lig4-deficient developing lymphocyte and neuronal cells.

Recent developments in enhancing the efficiency of CRISPR/Cas9- mediated knock-in in animals

Gene-editing technology can artificially modify genetic material of targeted loci by precise insertion, deletion, or replacement in the genomic DNA. In recent years, with the developments of zinc-finger endonuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats/CRISPR- associated protein 9 (CRISPR/Cas9) technologies, such precise modifications of the animal genomes have become possible. Although gene-editing tools, such as CRISPR/Cas9, can efficiently generate double-strand breaks (DSBs) in mammalian cells, the homology-directed repair (HDR) mediated knock-in (KI) efficiency is extremely low. In this review, we briefly describe the current development of gene-editing tools and summarize the recent strategies to enhance the CRISPR/Cas9- mediated KI efficiency, which will provide a reference for the generation of human disease models, research on gene therapy and livestock genetic improvement.

Gene editing technology and its research progress in China

Gene editing is a technology for precision modification of genome sequences or expressing transcripts. Early versions of such techniques include homing endonuclease, zinc finger endonuclease and transcription-like activator effectors. The CRISPR/Cas9 system has recently emerged as a powerful and versatile gene editing tool for a broad spectrum of applications. In this review, we discuss the recent development and applications of gene editing technology, and particularly focus on the progress in China, with the emphasis on mechanistic studies, development of new and extended technologies, the applications in animals, plants and the therapeutics.

153. Efficient Expression of mRNA PEG-Peptide Polyplexes in Mouse Liver

The delivery of mRNA has garnered much attention in recent years as genome editing techniques such as CRISPR/Cas9, Zinc Finger endonucleases, and TALEN endonucleases have been developed. The short-term protein expression offered by mRNA is attractive in these situations, where long-term expression could cause unwanted side-effects. mRNA also has the chief advantage of not needing to enter the nucleus to produce protein, a major barrier to successful nonviral DNA delivery. However, mRNA is highly susceptible to degradation by nucleases, and methods must be developed to stabilize it in the bloodstream before it can used effectively in vivo. PEGylated polyacridine peptides1 have been shown to protect plasmid DNA in the bloodstream for up 12 hours through a combination of nuclease protection and scavenger receptor inhibition2, and the protected DNA was shown to remain transfection competent by the delayed hydrodynamic dosing of normal saline. In this study, PEGylated polyacridine peptides were used to form stabilized luciferase mRNA polyplexes. In vitro transcription of a codon optimized mRNA construct containing both a 5’ and 3’ β-globin untranslated regions (UTRs). This mRNA was found to be 2000-fold more efficient at expressing luciferase in mouse liver when delivering a 1 mg dose hydrodynamically compared to mRNA lacking UTRs. The gene transfer efficiency in mouse liver increased an additional 15-fold when HD-dosing 1 mg of UTR-mRNA polyplex. The combined use of UTR-mRNA and PEGylated polyacridine peptide increased the expression efficiency in liver 3500-fold over the only prior report of hydrodynamically delivered mRNA in liver3. PEGylated polyacridine peptide, but not a simple polylysine PEG-peptide lacking acridine groups, was able to protect UTR-mRNA polyplexes from direct serum digestion for up to 30 minutes, demonstrating that a PEGylated polyacridine peptide binds to and stabilizes double stranded regions in native mRNA.

Erythrocytes from GGTA1/CMAH knockout pigs: implications for xenotransfusion and testing in non-human primates.

Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 galactose (aGal) and N-glycolylneuraminic acid (Neu5Gc). We have recently targeted the GGTA1 and CMAH genes with zinc finger endonucleases resulting in double knockout pigs that no longer express aGal or Neu5Gc and attract significantly fewer human antibodies. In this study, we characterized erythrocytes from domestic and genetically modified pigs, baboons, chimpanzees, and humans for binding of human and baboon natural antibody, and complement-mediated lysis. Distribution of anti-Neu5Gc IgG and IgM in pooled human AB serum was analyzed by ELISA. Erythrocytes from domestic pigs (Dom), aGal knockout pigs (GGTA1 KO), aGal and Neu5Gc double knockout pigs (GGTA1/CMAH KO), baboons, chimpanzees, and humans were analyzed by flow cytometry for aGal and Neu5Gc expression. In vitro comparative analysis of erythrocytes was conducted with pooled human AB serum and baboon serum. Total antibody binding was accessed by hemagglutination; complement-dependent lysis was measured by hemolytic assay; IgG or IgM binding to erythrocytes was characterized by flow cytometry. The pooled human AB serum contained 0.38 μg/ml anti-Neu5Gc IgG and 0.085 μg/ml anti-Neu5Gc IgM. Both Gal and Neu5Gc were not detectable on GGTA1/CMAH KO erythrocytes. Hemagglutination of GGTA1/CMAH KO erythrocytes with human serum was 3.5-fold lower compared with GGTA1 KO erythrocytes, but 1.6-fold greater when agglutinated with baboon serum. Hemolysis of GGTA1/CMAH KO erythrocytes by human serum (25%) was reduced 9-fold compared with GGTA1 KO erythrocytes, but increased 1.64-fold by baboon serum. Human IgG binding was reduced 27-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but markedly increased 3-fold by baboon serum IgG. Human IgM binding was decreased 227-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but enhanced 5-fold by baboon serum IgM. Removal of aGal and Neu5Gc antigens from pig erythrocytes significantly reduced human preformed antibody-mediated cytotoxicity but may have complicated future in vivo analysis by enhancing reactivity from baboons. The creation of the GGTA1/CMAH KO pig has provided the xenotransplantation researcher with organs and cells that attract fewer human antibodies than baboon and our closest primate relative, chimpanzee. These finding suggest that while GGTA1/CMAH KO erythrocytes may be useful for human transfusions, in vivo testing in the baboon may not provide a direct transplantation to the clinic.

TALE nucleases as a new tool for genome editing

Introducing targeted changes in the genome of living cells or whole organisms makes it possible to solve many problems of basic science, biotechnology, and medicine. Target gene knockout in zygotes helps to study the functions of the gene in the corresponding organisms, while replacement of single nucleotide in DNA provides an opportunity to correct gene mutations and to treat hereditary disorders. Adding a gene into a proper genome region can be used to construct producer cells or organisms with certain properties. Such genomic manipulations are possible due to the technology known as genome editing. In this technology, a break is introduced into a certain chromosomal DNA region with an endonuclease recognizing a unique sequence, and DNA integrity is then restored by cell repair systems. Custom-designed endonucleases able to cleave a selected target sequence are necessary tools for genome editing. Programmable endonucleases of a new type were constructed on the basis of bacterial transcription activator-like (TAL) effectors (TALEs), marking an important step in the development of genome editing and promoting its broad application. The review considers the history of discovering TALEs and creating TALE nucleases and describes their advantages over zinc finger endonucleases, which were constructed earlier. A section focuses on the genetic modifications that can be performed using various genome editing techniques.

Characterization of Defects in Ion Transport and Tissue Development in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-Knockout Rats

Animal models for cystic fibrosis (CF) have contributed significantly to our understanding of disease pathogenesis. Here we describe development and characterization of the first cystic fibrosis rat, in which the cystic fibrosis transmembrane conductance regulator gene (CFTR) was knocked out using a pair of zinc finger endonucleases (ZFN). The disrupted Cftr gene carries a 16 base pair deletion in exon 3, resulting in loss of CFTR protein expression. Breeding of heterozygous (CFTR+/−) rats resulted in Mendelian distribution of wild-type, heterozygous, and homozygous (CFTR−/−) pups. Nasal potential difference and transepithelial short circuit current measurements established a robust CF bioelectric phenotype, similar in many respects to that seen in CF patients. Young CFTR−/− rats exhibited histological abnormalities in the ileum and increased intracellular mucus in the proximal nasal septa. By six weeks of age, CFTR−/− males lacked the vas deferens bilaterally. Airway surface liquid and periciliary liquid depth were reduced, and submucosal gland size was abnormal in CFTR−/− animals. Use of ZFN based gene disruption successfully generated a CF animal model that recapitulates many aspects of human disease, and may be useful for modeling other CF genotypes, including CFTR processing defects, premature truncation alleles, and channel gating abnormalities.

Targeted DNA Mutagenesis for the Cure of Chronic Viral Infections

Human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), and herpes simplex virus (HSV) have been incurable to date because effective antiviral therapies target only replicating viruses and do not eradicate latently integrated or nonreplicating episomal viral genomes. Endonucleases that can target and cleave critical regions within latent viral genomes are currently in development. These enzymes are being engineered with high specificity such that off-target binding of cellular DNA will be absent or minimal. Imprecise nonhomologous-end-joining (NHEJ) DNA repair following repeated cleavage at the same critical site may permanently disrupt translation of essential viral proteins. We discuss the benefits and drawbacks of three types of DNA cleavage enzymes (zinc finger endonucleases, transcription activator-like [TAL] effector nucleases [TALENs], and homing endonucleases [also called meganucleases]), the development of delivery vectors for these enzymes, and potential obstacles for successful treatment of chronic viral infections. We then review issues regarding persistence of HIV-1, HBV, and HSV that are relevant to eradication with genome-altering approaches.

DNA Ligase III Promotes Alternative Nonhomologous End-Joining during Chromosomal Translocation Formation

Nonhomologous end-joining (NHEJ) is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4), suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ) pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1.

274. Correction of Albinism Mutations by Targeted Genomic Rearrangements Using Engineered Zinc Finger Endonucleases

274. Correction of Albinism Mutations by Targeted Genomic Rearrangements Using Engineered Zinc Finger Endonucleases

Gene targeting in plants: fingers on the move

Zinc-finger endonucleases (ZFNs) make targeted double-stranded breaks in genomic DNA and, thus, stimulate recombination and repair processes at specific sites. ZFNs can now be harnessed to stimulate homologous recombination and gene targeting in plants, which represents a major step towards modifying the plant genome more precisely. ZFN-mediated gene targeting is likely to become a powerful tool for genome research and genetic engineering.

Gene Correction in Human Cells Is Promoted by the Bacteriophage Protein Redβ.

Correction of a disease gene mutation by homologous recombination (HR) with a transfected DNA repair template represents an ideal form of gene therapy for many inherited hematological disorders. Therapeutic gene correction via HR in hematopoietic stem cells is unlikely to succeed, however, without major improvements in its efficiency. One promising approach is to use an engineered zinc finger endonuclease to specifically cleave the target gene and so promote HR between a dsDNA template and its chromosomal target (Urnov et al (2005) Nature 435:646-51). We are exploring an alternative approach based on the expression of Redβ, a single stranded (ss) DNA annealing protein from bacteriophage λ. In bacteria, Redβ promotes HR-mediated gene modification using templates with short regions of homology (e.g. 30–100nt). Such HR is independent of RecA-mediated host cell HR. For double stranded (ds) DNA templates, co-expression of λ exonuclease (the red α gene product) is required, but ssDNA templated modifications are particularly efficient and require only Redβ expression. Such properties could be useful for therapeutic gene correction if reproduced in human cells. Preliminary experiments in mouse embryonic stem cells suggested that Redβ can function in mammalian cells (Zhang Y. et al, (2003) BMC Mol. Biol. 4). To explore this further, we have generated a human cell line in which large amounts of Redβ can be expressed inducibly in the nucleus. In contrast to the over-expression of endogenous HR proteins Rad51 (RecA homologue) or Rad52 (ssDNA annealing protein), sustained Redβ over-expression is well tolerated, with no impairment of cell proliferation or viability. We find that Redβ does indeed promote HR in human cells. Surprisingly, dsDNA and ssDNA templated gene modification are stimulated to similar extents by Redβ. Stimulations are modest, but similar to those achieved by human Rad51 over-expression. The effects of co-expressing Red α are under investigation. We have also shown that ssDNA-templated gene correction is stimulated by transient, RNAi-mediated, depletion of the mismatch repair protein MSH2, and that such stimulation is additive with Redβ-stimulated gene correction.