• These nucleases can create double strand breaks (DSBs) in the DNA sequence that can be repaired by two main mechanisms: Non-homologous end joining (NHEJ), which introduces indels to modify the DNA sequence, and Homology-directed repair (HDR), which uses a donor template for accurate genetic correction. (uc3m.es)
  • Eukaryotic cells repair the DSBs by the fast but potentially mutagenic Non-Homologous End Joining (NHEJ), and by the accurate Homology Directed Repair (HDR) pathways. (europa.eu)
  • Usually, it is the NHEJ that repairs the DSBs. (europa.eu)
  • The resulting DNA double-strand breaks (DSBs) are repaired either by the error-prone nonhomologous end joining (NHEJ) or homology-directed repair ( 11 ). (life-science-alliance.org)
  • In contrast, NHEJ ligates the two broken ends of the DNA without a donor template, often resulting in random insertions or deletions (indels) that can disrupt coding sequences at the target site (for review see reference 14 ). (life-science-alliance.org)
  • Homology Directed Repair (HDR) or Non-homologous End-Joining (NHEJ), resulting in targeted integration or gene disruptions. (rroij.com)
  • NHEJ-mediated repair, on the other hand, is prone to mistakes because it results in the effective production of gene insertion or deletion (indels) of various lengths at the DSB site, eventually leading to gene inactivation. (rroij.com)
  • NHEJ can be used to inactivate a single or several genes in immortalized cell lines, but by causing loss-of-function mutations, it results in irreversible gene inactivation. (rroij.com)
  • Cas9-induced double-stranded breaks (DSBs) are generally repaired by one of two major pathways: non-homologous end joining (NHEJ) and homology-directed repair (HDR). (conferencedequebec.org)
  • The NHEJ pathway is usually characterized by imprecise re-joining of genomic DNA ends resulting in the creation of variable-length insertions and deletions (indels) at the DSB site. (conferencedequebec.org)
  • DSBs are one of the most deleterious DNA lesions, which are repaired by non-homologous end joining (NHEJ), homologous recombination (HR) or, if compromised, micro-homology mediated end joining (MMEJ). (crick.ac.uk)
  • Two main fix systems have got progressed to fix DSBs: NHEJ and homologous recombination (Human resources) structured fix (3,4). (cgp60474.com)
  • The majority of DSBs in a cell are repaired by non-homologous end-joining (NHEJ), which ligates blunt ends in a sequence-independent manner and can result in small insertions or deletions (indels) [ 1 ] . (encyclopedia.pub)
  • While NHEJ is the predominant pathway for DSB repair, its requirement for blunt ends renders it incapable of dealing with resected ends or one-ended DSBs resulting from replication fork collapse. (encyclopedia.pub)
  • In an HR-deficient context, DSBs that cannot be repaired by NHEJ must be channeled through error-prone homology-mediated pathways such as single-strand annealing (SSA) and alternative end-joining (Alt-EJ). (encyclopedia.pub)
  • Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. (aber.ac.uk)
  • The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ. (aber.ac.uk)
  • Although our cells are continuously challenged by DNA harm that's commonly fixed by nonhomologous end-joining (NHEJ) if not really properly repaired they PIK3R2 are able to lead in some instances to complicated chromosomal translocations cell loss of life and tumor (1 2 Although last could be a uncommon event cells harboring mutations which result in uncontrolled cell routine have got a selective benefit and therefore might trigger tumors. (healthandwellnesssource.org)
  • Two main repair mechanisms have got PF-3845 evolved to correct DSBs: NHEJ and homologous recombination (HR) structured fix (3 4 NHEJ can result in an inaccurate fix as it might involve processing from the DNA ends at the website of damage. (healthandwellnesssource.org)
  • Chromosomal double-strand breaks (DSBs) in mammalian cells are repaired by either homology-directed repair (HDR), using a homologous sequence as a repair template, or nonhomologous end-joining (NHEJ), which often involves sequence alterations at the DSB site. (arizona.edu)
  • In the presence of an HR Donor with homology to the region adjacent to the DSB (blue areas of the genomic and vector DNA) homologous recombination (HR) is favored over non-homologous end joining (NHEJ). (reportergene.com)
  • We propose a model in which the ends of different, Top1-associated DSBs are joined via NHEJ, which results in deletion of the intervening sequence. (duke.edu)
  • 53BP1 (605230) is critical for the control of double-strand break (DSB) repair, promoting nonhomologous end-joining (NHEJ) and inhibiting the 5-prime end resection needed for homology-directed repair (HDR). (wikisequences.org)
  • Two main mechanisms for intron loss have been proposed, that is reverse transcription (RT)-mediated intron loss and intron deletion triggered by repair of double strand breaks (DSBs) via non-homologous end joining (NHEJ). (biomedcentral.com)
  • A signature of genomic deletion of introns via NHEJ is the presence of 2-8 bp of micro-homology. (biomedcentral.com)
  • They cause double-stranded breaks (DSBs), which can be repaired using one of two DNA repair pathways: homology directed repair (HDR) or non-homologous end joining (NHEJ). (experiome.com)
  • In the absence of a repair template, however, DSBs are repaired by the NHEJ pathway, which introduces insertions or deletions by editing the DNA region, causing target genes to be disrupted by moving the reading frame. (experiome.com)
  • Instead of the HDR, CRISPR/Cas nucleases-induced DSBs are largely repaired by the efficient eukaryotic cellular NHEJ mechanism. (experiome.com)
  • The two most commonly used pathways to repair DSBs in higher eukaryotes include non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is considered to be error-prone, intrinsically mutagenic quick fix remedy to seal together the broken DNA ends and restart replication. (springeropen.com)
  • Non-homologous end joining (NHEJ), which does not depend upon sequence homology, is the key repair pathway during the G0/G1 stages of the cell cycle [ 10 ]. (springeropen.com)
  • A second NHEJ concomitant pathway often referred to as alternative-NHEJ (Alt-NHEJ), also known as Microhomology-mediated end joining (MMEJ), is another well-studied pathway for repairing DSBs in DNA [ 16 ]. (springeropen.com)
  • Using the SURVEYOR nuclease assay 13 , we assessed the ability of each Cas9-sgRNA complex to generate indels in human embryonic kidney (HEK) 293FT cells through the induction of DNA doublestranded breaks (DSBs) and subsequent nonhomologous end joining (NHEJ) DNA damage repair (Online Methods). (cdc.gov)
  • These are extremely hazardous for a cell, because if left unrepaired, DSBs can have pathological consequences, such as cell death, or drive cells to genomic instability and tumor development. (nature.com)
  • If left unrepaired, DSBs can lead to cell death or if repaired incorrectly can result in chromosome rearrangements that drive cancer development. (crick.ac.uk)
  • If left unrepaired, DSBs can cause chromosomal aberrations, mutagenesis, and cell death, leading to the development of severe diseases including cancer [ 1 ] [ 2 ] . (encyclopedia.pub)
  • DSBs are cytotoxic lesions, which if left unrepaired could lead to genomic instability, cancer and even cell death. (springeropen.com)
  • DNA double-strand breaks (DSBs) are repaired by two main pathways: nonhomologous end-joining and homologous recombination (HR). Repair pathway choice is thought to be determined by cell cycle timing and chromatin context. (diagenode.com)
  • CRISPR-Cas9-based genome editing has transformed the life sciences, enabling virtually unlimited genetic manipulation of genomes: The RNA-guided Cas9 endonuclease cuts DNA at a specific target sequence and the resulting double-strand breaks are mended by one of the intrinsic cellular repair pathways. (life-science-alliance.org)
  • Given the cytotoxic nature of these lesions, the cell has developed multiple pathways of DSB repair, some which result in mutagenic repair, and others that use a template to ensure error-free repair. (encyclopedia.pub)
  • 2013 ). Interestingly, the molecular mechanisms underlying the patterning of meiotic DSBs are quite distinct in different species with apparently two major pathways. (springer.com)
  • Cells have evolved with conserved recombination mediated genome editing pathways as a mean for repairing DSBs and restarting replication forks, thus allowing genome duplication to continue [ 8 ]. (springeropen.com)
  • DNA double strand break repair (DSBR) pathways are generally classified based on whether sequence homology is used to join the broken DNA ends. (springeropen.com)
  • Cas9 has intrinsic nuclease activity, which causes sequence specific DNA double-strand breaks (DSBs), leading to activation of cellular pathways such as homology dependent repair (HDR) for gene conversion. (technologypublisher.com)
  • By recruiting the catalytic subunit of DNA-protein kinase (DNA-PKcs, also known as PRKDC ) and other proteins, Ku enables the repair of DNA DSBs through the non-homologous end joining pathway. (nature.com)
  • Recent studies indicate that CRISPR-induced DNA cuts may also result in the exchange of genetic information between homologous chromosome arms. (life-science-alliance.org)
  • Double strand breaks are then repaired via homologous recombination or non-homologous end-joining, resulting in indels (insertion or deletion of bases in the genome) of variable length. (vectorbuilder.kr)
  • The donor vector is designed with the desired insertion sequence flanked by target site specific upstream and downstream homologous sequences to facilitate efficient recombination following CRISPR-generated DSBs. (vectorbuilder.kr)
  • Homologous recombination (HR) plays a critical role in largely error-free repair of mitotic and meiotic DNA double-strand breaks (DSBs). (crick.ac.uk)
  • Because of this configuration, on-target integration that results from homologous recombination will not include the PGK-hsvTK cassette-only randomly-integrated off-target events will lead to integration of PGK-hsvTK and resulting TK activity. (reportergene.com)
  • Ku exists as a KU70 (also known as XRCC6 )-KU80 (also known as XRCC5 ) heterodimer that binds the ends of DNA double-strand breaks (DSBs). (nature.com)
  • PARP-1 is rapidly recruited and activated by DNA double-strand breaks (DSBs). (nature.com)
  • The production of nuclease-induced Double-Stranded Breaks (DSBs) leads to the stimulation of very effective cellular DNA recombination processes in mammalian cells, which leads to targeted DNA changes. (rroij.com)
  • We have discovered that elevated levels of the metabolites 2-hydroxyglutarate (2HG), succinate, and fumarate in human cancers suppress homology-dependent repair (HDR) of DNA double-strand breaks (DSBs). (nih.gov)
  • All of this is possible without double strand breaks (DSBs) or donor DNA templates. (addgene.org)
  • Double-strand breaks (DSBs) are the greatest threat to genomic integrity and can result from endogenous sources such as reactive oxygen species (ROS) and replication stress or exogenous insults such as UV, ionizing radiation (IR), and chemotherapeutic compounds. (encyclopedia.pub)
  • DNA double strand breaks (DSBs) occur constantly in eukaryotes. (aber.ac.uk)
  • INTRODUCTION The most unfortunate kind of DNA harm is certainly DNA double-strand breaks (DSBs). (healthandwellnesssource.org)
  • In the 1980s, Mario Capecchi 1st founded gene-targeting CAB39L technology, along with the concept of harnessing double-strand breaks (DSBs) for genome editing (Capecchi, 1980). (baxkyardgardener.com)
  • The double-stranded breaks (DSBs) launched by CRISPR/Cas or TALEN complexes stimulates the endogenous DNA restoration machineries, nonhomologous. (baxkyardgardener.com)
  • Double-strand breaks (DSBs), one class of the most harmful DNA damage forms that bring elevated health risks, need to be repaired timely and effectively. (jesc.ac.cn)
  • These genes function as caretakers in the maintenance of genomic stability, in part, by participating in homology-directed DNA repair (HDR), an error-free mechanism for the repair of double-strand breaks (DSBs). (johnshopkins.edu)
  • Meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs), generated by SPO11 and accessory proteins (de Massy 2013 ). (springer.com)
  • Recent studies revealed classes of recurrent DNA double-strand breaks (DSBs) in neural stem/progenitor cells, including transcription-associated, promoter-proximal breaks and recurrent DSB clusters in late-replicating, long neural genes that may give rise to somatic brain mosaicism. (stanford.edu)
  • The genome is under constant assault from a multitude of sources that can lead to the formation of DNA double-stand breaks (DSBs). (springeropen.com)
  • DNA double strand breaks (DSBs) result as a consequence of the disassembly of the DNA double helix leading to the disruption of the stability of the genome. (springeropen.com)
  • Regardless of how DSBs are formed, faithful repair of these breaks are absolutely essential for maintenance of genome integrity. (springeropen.com)
  • Ionizing radiation is well known to induce oxidative DNA damage, such as DNA double-strand breaks (DSBs), and consequently trigger the DNA damage response (DDR), including cell cycle arrest and apoptosis. (researchsquare.com)
  • This is due to a paucity of functional annotation from sequence homology, which is compounded by low genetic tractability compared with many model organisms. (portlandpress.com)
  • For refined and precise genome editing purposes, homology-directed repair is harnessed to copy a specific DNA template (single-stranded or double-stranded) into the target site ( 2 , 9 , 12 , 13 ). (life-science-alliance.org)
  • The engineering of unique Zinc-Finger Nucleases (ZFNs) or mega nucleases has been the focus of study in the early stages of genome editing to induce the desired DSBs at specific DNA target sites. (rroij.com)
  • Inhibition of KDM4B results in broad H3K9me3 hypermethylation across the genome, masking the H3K9me3 chromatin signal at the DSB that is essential for recruitment of TIP60 and other factors to initiate HDR. (nih.gov)
  • Meiotic DSBs are not randomly distributed within the genome but mostly occur at discrete regions, called hotspots (Baudat et al. (springer.com)
  • Hence, although the repair of DSBs is crucial for the maintenance of genome integrity the process of repair need to be well regulated and closely monitored. (springeropen.com)
  • These results suggest that HR can be templated in cis and suggest a role for chromosomal context in the maintenance of NOR genomic stability. (diagenode.com)
  • This can result in replacement of the target genomic DNA sequence with the sequence from the donor vector containing desired mutation(s) or fragments to be knocked in. (vectorbuilder.kr)
  • The pegRNA is a guide RNA that also encodes the RT template, which includes the desired edit and homology to the genomic DNA locus. (addgene.org)
  • Furthermore, DSBs in cells caused by DNA repair mechanisms are reported, which result in a variety of undesirable genomic changes such as significant deletions and translocations. (experiome.com)
  • Taken together, these results indicate that ΔNp63α represses p53-related radiation-induced DDR, thereby potentially causing genomic instability in epithelial stem cells. (researchsquare.com)
  • The ability of CRISPR-Cas9 to introduce several concurrent DSBs at defined positions has enabled engineering of tumor-associated chromosomal translocations resembling those observed in cancers, and hence to establish and test novel in vitro and in vivo tumor models ( 2 , 23 , 24 , 25 ). (life-science-alliance.org)
  • DSBs can be repaired by homology-directed repair (HDR) using exogenous donor DNA template, which is co-introduced into cells with the CRISPR/Cas9 components. (vectorbuilder.kr)
  • Second, a single CRISPR-Cas9-based plasmid carrying a highly specific single guide RNA (sgRNA) targeting crtMN operon and a changeable editing template was constructed to facilitate simultaneous insertion of multiple gene-copies through homology-directed repair (HDR). (biomedcentral.com)
  • However, the conventional CRISPR technology depends on DSBs which may lead to undesired genetic events such as off-target insertions, deletions, and chromosomal translocations. (technologypublisher.com)
  • 2012). By generating more than one DSB, it becomes possible to produce even more types of changes, including chromosomal deletions, gene inversions and, with DSBs on two different chromosomes, chromosomal translocations (Morgan et al. (baxkyardgardener.com)
  • However, erroneous repair of DSBs can lead to chromosomal rearrangements and loss of heterozygosity, which in turn can also cause cancer and cell death. (springeropen.com)
  • Failure to repair DSBs can lead to unwanted consequences, such as loss of genetic information, chromosomal rearrangements and even cell death. (springeropen.com)
  • Here, we show that in Drosophila , the detected Cas9-mediated editing events frequently resulted in germline-transmitted exchange of chromosome arms-often without indels. (life-science-alliance.org)
  • They found that orientation matters: fusing the RT to the C-terminus of the Cas9 nickase resulted in higher editing efficiency. (addgene.org)
  • Even though gene knock-outs can result from DSBs caused by Cas9 alone, SBI recommends the use of HR targeting vectors (also called HR donor vectors) for more efficient and precise mutation. (reportergene.com)
  • Why an HR targeting vector is a recommended Even though gene knock-outs can result from DSBs caused by Cas9 alone, SBI recommends the use of HR targeting vectors (also called HR donor vectors) for more efficient and precise mutation. (reportergene.com)
  • The HDR pathway can be used to precisely repair a DSB in the current presence of an exogenously added DNA donor template that bears homology towards the DNA sequences upstream and downstream of the mark site. (conferencedequebec.org)
  • The donor vector contains the desired insertion sequence flanked by upstream and downstream homology arms. (vectorbuilder.kr)
  • Alt-EJ operates by an analogous mechanism, only it requires a shorter length of resection and anneals regions of microhomology, resulting in smaller deletions. (encyclopedia.pub)
  • The majority of repair events resulted in no loss of sequence and small (1-20 bp) deletions occurred at a minority (25-45%) of repair junctions. (aber.ac.uk)
  • In the current study, we report a type of DSB-dependent mutation associated with Top1cc stabilization: large deletions (median size, ∼100 bp) with little or no homology at deletion junctions. (duke.edu)
  • Our previous work indicated that the formation of unsaturated RecA nucleofilaments plays an essential role in homology recombination (HR) pathway which can accurately repair DSBs. (jesc.ac.cn)
  • DSBs not only ensue from normal cellular metabolism, in the form of reactive oxygen species that can oxidize DNA bases [ 1 , 2 ], but can also be generated during physiological processes like chromosome replication, meiotic recombination and DNA replication transcription collision [ 3 - 7 ]. (springeropen.com)
  • We discuss what factors determine the sub pathway choice including etiology of the DSB, chromatin structure at the break site, processing of the DSBs and the mechanisms regulating the sub-pathway choice. (springeropen.com)
  • In practically all cell types and organisms, nuclease-induced DNA DSBs can be repaired by one of two basic mechanisms. (rroij.com)
  • Overexpression of exogenous BCL2 in a lung cancer cell line that expresses no detectable level of the endogenous protein resulted in decreased DNA DSB repair, as shown by persistent DNA DSBs (shown using pulsed field gel electrophoresis) and γH2AX foci, and increased levels of cytogenetic abnormalities compared with controls after exposure to ionizing radiation (IR). (nature.com)
  • Lastly, knockdown studies revealed that the depletion of endogenous Akt1 and Akt3, but not Akt2, inhibit clonogenic activity and repair of ionizing radiation (IR)induced DNA DSBs, top to radiosensitization. (bet-bromodomain.com)
  • Different pharmacological, cellular and genetic approaches have been tried showing promising results, but, until now, treatment has focused primarily on symptomatic relief such as daily wound care or pain reduction. (uc3m.es)
  • Targeted nucleases have been developed as an alternate strategy to increasing the efficacy of HDR-mediated genetic modification when it was recognized that DSBs could increase the incidence of HDR by numerous orders of magnitude. (rroij.com)
  • In Saccharomyces cerevisiae , meiotic DSBs preferentially occur at nucleosome-depleted regions in gene promoters (Ohta et al. (springer.com)
  • Indeed, in the absence of PRDM9, meiotic DSBs occur at transcription regulatory regions, such as promoters and enhancers (Brick et al. (springer.com)
  • PARP-1 plays a critical role in DSB sensing and we have shown that PARP-1 recruitment and activation occur within 100 ms after introduction of DSBs. (nature.com)
  • If indels exist in the coding sequence, frame shift mutations occur, resulting in mRNA degradation or the generation of non-functional truncated proteins via nonsense-mediated decay. (rroij.com)
  • The break is then repaired by the annealing of these complementary sequences, resulting in deletion of the intervening sequence [ 3 ] [ 9 ] . (encyclopedia.pub)
  • Importantly, DSBs within rDNA recruit the HR machinery throughout the cell cycle. (diagenode.com)
  • Importantly, the results will provide a base for a long-awaited revolution in personalized medicine. (europa.eu)
  • Although on the surface and in terms of oncogenesis these findings make sense, it seems counter-intuitive that a protein capable of increasing cell survival after exposure to DNA-damaging agents would also have the capacity to prevent the repair of DNA DSBs. (nature.com)
  • This mutation results in a premature termination codon (PTC) and when it is present in both COL7A1 alleles, it leads to the absence of this protein. (uc3m.es)
  • Protein expression attributed to a single copy of the integrated gene usually results in lower yields of product compared to the use of high copy-number vectors. (biomedcentral.com)
  • We investigated the HDR efficiency of inserting a green fluorescent protein (GFP) tag using either long, single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) templates (700 bp insert, 200 bp homology arms). (idtdna.com)
  • We released DSBs in populations of cells with equivalent kinetics and performance and processed through security for chemical substance substances that modulate DSB fix by HDR. (cgp60474.com)
  • Furthermore, these events accumulated in quiescent cells, suggesting that the causative DSBs may arise outside the context of replication. (duke.edu)
  • The mechanistic factors promoting these different classes of DSBs in neural stem/progenitor cells are not understood. (stanford.edu)
  • Together, these information indicate that Akt1 and Akt3, but not Akt2, physically interact with DNAPKcs, as a result stimulating the repair of DSBs and hence safeguarding KRASmut cells against IR. (bet-bromodomain.com)
  • Our results, therefore, place PARP-1 activation as a critical early event for DNA DSB repair activation and regulation of resection. (nature.com)
  • Targeted introduction of DSBs into rDNA, but not abutting sequences, results in ATM-dependent inhibition of their transcription by RNA polymerase I. This is coupled with movement of rDNA from the nucleolar interior to anchoring points at the periphery. (diagenode.com)
  • Remarkably, inhibition of PARP-1 leads to hyperresected DNA DSBs. (nature.com)
  • RIF1 inhibition resulted in radiosensitivity and a defect in the intra-S-phase checkpoint. (wikisequences.org)
  • Homology phosphorylated form( HDR) through environmental particle enhancing( phagocytosis), uncertain to HDR through separate efficiency cholesterol( HRR), is individual entrepreneur of DNA crucial state degradation is( DSBs), comprised by 1-diphosphate rimantadine and concentration of the complete lacking intermediate coupled factors( IRIF) at DNA DSB members. (familie-vos.de)
  • As a result, mutation repair or novel sequence insertion occurs in a site-specific way. (rroij.com)
  • Following its decision, the EU Court considers that the NBTs operate "at a rate out of all proportion to those resulting from the application of conventional methods of mutagenesis. (frontiersin.org)
  • In order to initiate HR, the DSB is resected, leaving a segment of single-stranded DNA (ssDNA) that is coated first by RPA, then by RAD51 in order to facilitate homology search and invasion of the sister chromatid-a process known as strand exchange. (encyclopedia.pub)
  • Our results suggest that the integrity of normally sealed esophageal epithelia is compromised in the EoE patients compared to control subjects, and this is due to alterations in the expression of cell adhesion molecules at the esophageal epithelium. (bvsalud.org)
  • The uncommon incidence of HDR may result in a much less accurate DSB fix and is certainly most likely the cause for low performance of gene concentrating on utilized in analysis and disease therapy. (cgp60474.com)
  • Homology described fix (HDR) is certainly regarded accurate, since it relies on the sis chromatid as a template, and can as a result, end up being employed during or after S-phase of the cell routine generally. (cgp60474.com)
  • Homology directed fix (HDR) is known as accurate because it depends on the sister chromatid being a template and will therefore mainly be used during or after S-phase from the cell routine. (healthandwellnesssource.org)
  • Recombinant antibodies are highly consistent from lot to lot, thereby ensuring reproducible experimental results. (cellsignal.jp)
  • DNA double-strand fractures (DSBs) are the most serious type of DNA harm. (cgp60474.com)
  • A Ku-modulated two-ended versus one-ended chromosome break model is presented to explain these results. (arizona.edu)
  • 2010 ). PRDM9 includes several functional domains, such as a DNA-binding zinc finger array, a histone lysine methyltransferase PR/SET (PRDI-BF1 and RIZ1 homology) domain, and a Krüppel-associated box (KRAB)-related domain. (springer.com)
  • Indel mutations can disrupt the translational Nitrarine 2HCl reading frame and therefore if introduced into Nitrarine 2HCl coding sequence may result in knockout of the target gene. (conferencedequebec.org)
  • In addition, HR donors can include up to 6-8 kb of open reading frame for gene knock-ins or tagging, and, when small mutations are included in either 5' or 3' homology arms, can make specific, targeted gene edits. (reportergene.com)