Isolated T cells were cultured at 1million cells per ml in ImmunoCult medium (STEMCELL) with 5% FBS (Bio-Techne), 50M 2-mercaptoethanol (Sigma-Aldrich) and 10mM N-acetyl l-cysteine (Sigma-Aldrich) and were stimulated for 2d before electroporation with anti-human CD3/CD28 magnetic dynabeads (Thermo Fisher Scientific) at a beads to cells concentration of 1:1, along with a cytokine cocktail of IL-2 at 200Uml1 (STEMCELL), IL-7 at 5ngml1 (STEMCELL) and IL-15 at 5 ngml1 (STEMCELL). Lentiviral constructs encoding gRNAs targeting FANCA, FANCD2, FANCF, FANCJ, FANCM, 53BP1, NEIL3, XPC, XPF, POLB, TRAIP, XRCC1 or a nontargeting sequence (Supplementary Data) were separately transduced into K562 cells containing dCas9KRAB (clone K1e14). Meanwhile, from the beginning, every piece of single stranded DNA is covered by the protein RPA (Replication Protein A). 2c), which are useful cells for regenerative medicine applications. Cells were then flowed at 96 and 120h post electroporation to measure editing. A. Exact P values in the bottom right plot (from left to right) in orange are 0.7903, 0.4724 and 0.8799, and in blue, 0.7229, 0.8458 and 0.6024. d, xHDRT activity requires components of the FA pathway. ICLs added to an HDRTwhich we refer to as xHDRTsdramatically improve editing rates in nonviral gene editing workflows in a dose-dependent manner (Fig. Sci. Your favorite. In the budding yeast Saccharomyces cerevisiae homology directed repair is primarily a response to spontaneous or induced damage that occurs during vegetative growth. Source data are provided with this paper. Source Data Extended Data Figs. This is apparent during fluorescent tagging of endogenous genes, where we observe an approximately threefold increase in GFP cells rather than any decrease caused by frame- or codon-disrupting variants in the GFP donor sequence (Fig. We therefore conclude that xHDRTs specifically boost HDR frequency rather than altering global DNA repair preferences. 50, 11321139 (2018). For example, a receptor of the cell that can receive a signal to stop dividing may malfunction, so the cell ignores the signal and keeps dividing and can form a cancer. The resulting cell populations were selected for homogeneity using puromycin (1gml1). Correlations between indel spectra for pairwise comparisons were calculated using Pearson correlations (seaborn v 0.12.0). PCR2 products were SPRI cleaned, quantified by Qubit, normalized and pooled at equimolar amounts. HDR preferably occurs through the error-free . Homologous recombination, also known as homology-directed repair (HDR), is the predominant pathway to repair DSBs in an error-free manner. Unlike in DSBR, following strand invasion and D loop formation, the newly synthesized portion of the invasive strand is displaced from the template and returned to the processed end of the non-invading strand at the other DSB end. (2023)Cite this article. of n=4 biological replicates; comparisons between xHDRT-edited samples versus HDRT-edited controls. All data were statistically analyzed using two-tailed t-tests. 43, 247258 (2015). A. Here we pave the way to increasing gene replacement efficiency by evaluating the effect of expression of multiple heterologous recombinases on intrachromosomal homologous recombination (ICR) in Nicotiana tabacum plants. This indicates that ICLs do not increase the nuclear abundance of xHDRTs relative to uncrosslinked templates. Data displayed as the means.d. Ran FA, et al. Doudna, J. The efficiency of homologous recombination is increased by crosslinking template DNA. Biotechnol. HMI-103 is a one-time, in vivo, nuclease-free gene editing candidate for PKU designed to harness the body's natural DNA repair process of homologous recombination to replace the disease-causing gene with a functional gene and liver-specific promoter and to maximize Exact p-values for HDRT and xHDRT calculated using an unpaired two-tailed T-test are respectively 0.0035 and 0.0033. All data are represented as the meanSD generated from n=3 biological replicates. These values were plotted on a per nucleotide basis (Extended Data Fig. H2AX is phosphorylated throughout the area surrounding the damage, not only precisely at the break. Optimizing the DNA Donor Template for Homology-Directed Repair of Double-Strand Breaks - ScienceDirect Volume 7, 16 June 2017, Pages 53-60 Original Article Optimizing the DNA Donor Template for Homology-Directed Repair of Double-Strand Breaks Fei Song 1 , Knut Stieger 1 Add to Mendeley Significance values are displayed above the experimental sample. HEK293T cells were cultured in DMEM media supplemented with 10% FBS, 1% sodium pyruvate and 100gml1 penicillinstreptomycin. 36, 95102 (2018). MRE11-RAD50-NBS1 is a critical regulator of FANCD2 stability and function during DNA double-strand break repair. The most common form of homology-directed repair is called homologous recombination (abbreviated HR), which has the longest sequence homology requirements between the donor and acceptor DNA. dHJs are four-stranded branched structures that form when elongation of the invasive strand captures and synthesizes DNA from the second DSB end. Onemillion stimulated T cells were debeaded for several minutes before nucleofection and pelleted at 500g for 3min. Chris D. Richardson. Interstrand crosslinking of homologous repair template DNA enhances gene editing in human cells. Proc Natl Acad Sci U S A. Due to the local effect of the ICL, we hypothesized that DNA repair factors recruited to the ICL might prime the xHDRT for use as a template. Ct values were calculated between the HDRT and xHDRT molecules after the pooling of biological triplicates. Exact p-values between treatments for the bottom left plot in orange from left to right are 0.40349, 0.39957, 0.23857 and in blue 0.01990, 0.03006, 0.72860. H.I.G. Theconservative methods are characterized by use of a homologous donor (e.g., sister chromatid, plasmid, etc. We thank C. Arnold for assistance with iPSC protocols. iPSCs were then treated with 3ml prewarmed Accutase (Innovative Cell Technologies), and the vessel was incubated at 37C for 5min. To estimate the optimal number of ICLs per xHDRT, we developed a qPCR-based assay that approximates the number of crosslinks within a given DNA molecule (Extended Data Fig. 64, 91529159 (2004). 11, 2109 (2020). Hussmann, J. Homology-Directed Repair and the Role of BRCA1, BRCA2, and Related Proteins in Genome Integrity and Cancer Annu Rev Cancer Biol. All data were statistically analyzed using two-tailed t-tests. Western blots for phospho-Chk1, and phospho-Chk2, and phospho-DNA-PK 24hours after the indicated treatments. of at least n=2 biological replicates. Percent incorporation of a fluorophore at the LMNB1, IL2RA, or RAB11A loci in primary T cells. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in & Kim, J.-S. Lentivirus was produced by transfecting HEK293T cells with standard packaging vectors using the TransIT-LT1 transfection reagent (MIR 2306, Mirus Bio). Proc Natl Acad Sci 95: 5172-5177 [PMC free article] . Psoralen crosslink density is a function of the TA content of the DNA, the psoralen concentration and the UV dosage, and may thus vary between HDRTs. Commun. RNA was extracted using RNeasy Mini Kits (Qiagen). Foote, K. M. et al. Introduction One of the most severe types of DNA damage is a double-strand break (DSB), which can lead to chromosome rearrangement, chromosome loss, or even cell death. For routine passaging, at 70% confluency, old media was aspirated and cells were washed with 5ml room temperature DPBS before dissociation. iPSC RNPs were made before electroporation as described above. PLoS ONE 9(8); (2014). Data displayed as the means.d. 13 and Extended Data Figs. All relevant code supporting the findings of this study are available in the Supplementary Information section. 7, 93 (2015). Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. 4 xHDRTs do not increase mutation frequency at edited loci. Cts were averaged between two concentrations of input DNA. Cell 82, 348388 (2022). We acknowledge the assistance of J. Smith (manager of the BNL) and the use of the Biological Nanostructures Laboratory within the California NanoSystems Institute, supported by the University of California, Santa Barbara, and the University of California, Office of the President. 6e). Reaction mixtures were electroporated in 4D Nucleocuvettes (Lonza) and later recovered to culture dish wells containing prewarmed media. Furthermore, the requirement for ATR activity during xHDRT editing indicates that this may be a mechanistically distinct form of recombination. Want to know more? 7b,c). Here we report that adding ICLsa substrate of the FA pathwayto donor DNA in gene editing reactions dramatically enhances the frequency with which the template is used in HDR. A technique that pushes the size limitations of ssODNs is Easi-CRISPR (Quadros et al., 2017). The Road Less Traveled: Strategies to Enhance the Frequency of Homology-directed Repair (HDR) for Increased Efficiency of CRISPR/Cas-mediated Transgenesis (2018). 3d) by approximately threefold compared to uncrosslinked templates (Fig. This protein error may cause processes in the cell to fail. After standard nucleofection, cells (K562s or T cells) were recovered in media containing the indicated concentration of ATR inhibitor (AZ20 or Ceralasertib), ATM inhibitor (KU55933) or DNA-PK inhibitor (NU7026). DPBS was gently aspirated from the T cell pellet and then resuspended in 15l of buffer P3 (Lonza). During this period of arrest, oocytes are subject to spontaneous DNA damage including double-strand breaks. DPBS (2ml) was added and lifted cells were triturated, followed by the addition of another 3ml DPBS for a final wash. Data displayed as the means.d. Homologous recombination is an effective way to generate recombinant viruses for vaccine research such as pseudorabies virus (PRV) and adenovirus. Adli, M. The CRISPR tool kit for genome editing and beyond. Li, K et al. 4a, unedited). Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Other forms of homology-directed repair include single-strand annealing (abbreviated SSA) and breakage-induced replication, and these require shorter sequence homology relative to HR ( 4 , 5 ). Iyama, T. et al. Significance values are displayed above the experimental sample, *P0.05, **P0.01, ***P0.001, ****P0.0001; NS, not significant. and C.D.R. Nguyen, D. N. et al. RNPs were made before electroporation as described above. We describe a strategy to boost the efficiency of gene editing via homology-directed repair (HDR) by covalently modifying the template DNA with interstrand crosslinks. (d) xHDRT activity is partially dependent on FANCA. Canny, M. D. et al. Absolute yield of RAB11A-GFP positive, viable T cells achieved using either linear PCR-derived or plasmid HDRT/xHDRT when treated with titrated concentrations of AZ20 (ATR inhibitor). 2a). Mar 12, 2015; updated by Christina Mork, Jul 27, 2020; updated by Susanna Stroik January 24, 2023. In HR, recombination occurs between sister chromatids present . Immunol. Slider with three articles shown per slide. Media was aspirated, and cells were washed again with DPBS. The fertility of females and the health of potential offspring critically depend on an adequate availability of high quality oocytes. All cell line and primary cell work were approved by UCSB BUA2019-15. U2OS cells were cultured in DMEM supplemented with only 10% FBS and 100gml1 penicillinstreptomycin. This will serve as both a substrate for proteins required for strand invasion and a primer for DNA repair synthesis. Extended Data Fig. 2, 6 and 7, http://creativecommons.org/licenses/by/4.0/. Samples were vortexed for 10s at full speed, boiled for 8min and passed three times through a 25G needle. ^ In the literature, homologous directed repair (HDR) is also referred to as: homologous repair, homology recombination repair, knock-ins, homology-driven recombination, ssODN-mediated-homology-directed repair, ssODN-mediated gene repair and precise genome modification. Extended Data Fig. Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing. In the classical DSBR pathway, the 3 ends invade an intact homologous template to serve as a primer for DNA repair synthesis, ultimately leading to the formation of double Holliday junctions (dHJs). Recently, several developments have shed light on the DNA repair pathways that become engaged at ALT telomeres, implicating ALT telomeres as DNA repair hot spots. Beeke Wienert, David N. Nguyen, Jacob E. Corn, Tarun S. Nambiar, Pierre Billon, Alberto Ciccia, Chris D. Richardson, Katelynn R. Kazane, Jacob E. Corn, Holly A. Rees, Wei-Hsi Yeh & David R. Liu, Brett M. Sansbury, Amanda M. Hewes & Eric B. Kmiec, Shota Nakade, Keiji Mochida, Takashi Yamamoto, Zsolt Bodai, Alena L. Bishop, Alexis C. Komor, Tsuyoshi Fukushima, Yosuke Tanaka, Toshio Kitamura, Nature Biotechnology If material is not included in the articles Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Common crosslinking agents include psoralen, which crosslinks opposing thymines at TA sequences 23, and cisplatin, which crosslinks opposing guanines at GC sequences in dsDNA24. J. Chem. Making designer mutants in model organisms. The HDR mechanism has also been found in more simple organisms, such as yeast. Enoiu, M., Jiricny, J. Homology directed repair (HDR), a naturally occurring nucleic acid repair system, can be used to modify genomes in many organisms, including humans (Sander and Joung, 2014). Exact p-values calculated using an unpaired two-tailed T-test between treatments for the top left plot in orange from left to right are 0.63111, 0.04080, 0.03184 and in blue, 0.00128, 0.31019, 0.19713. H.I.G. dsDNA donor (1g), prepared at 1gl1, was then added to the RNP mixture. 7b). Introduction What does DNA have to do with cancer? Edited or unedited samples were amplified with primers described in Supplementary Data (oCR3775oCR3776 for HBB; oCR3807oCR3808 for RAB11A) using GoTaq master mix (Promega) and 200ng of gDNA. We archive and distribute high quality plasmids from your colleagues. In general, the insertion sites of the modification should be very close to the DSB, ideally less than 10 bp away if possible. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Percent of asynchronous cells edited with uncrosslinked donors or xHDRTs at the indicated point in the cell cycle. HDR is initiated by the presence of double strand breaks (DSBs) in DNA (Liang et al. For qPCR, between 2.5105 and 1106 CRISPRi cells were collected. While this pathway may not be immediately applicable in DSB-induced genome engineering, it has biological importance for the repair of breaks that lack a second end to enable DSBR or SDSA. 6b), indicating that xHDRTs act independently from the anionic polymer effect. PCR amplicons (PCR1) were purified using SPRI beads, run on a 1.0% agarose gel to validate size and quantified by Qubit. After three washes with PBS containing 1% BSA, cells were treated for 30min with RNase A and stained for 10min with propidium iodide and run on the flow cytometer. 24, 10121019 (2014). The supernatant was carefully aspirated, and cells were resuspended in 10ml fresh mTeSR1 containing ROCK inhibitor (ROCKi) and counted using a Countess slide. We also note that transcription itself is not required, as xHDRTs lacking any eukaryotic promoters support increased levels of HDR (Fig. Easi-CRISPR in vitro transcribes RNA encoding your repair template, then uses reverse transcriptase to generate the complementary ssDNA. Internet Explorer). The function of RPA is likely to keep the single stranded DNA pieces stable until the complementary piece is resynthesized by a polymerase. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Increased editing from xHDRTs is driven by events on the template molecule and requires ataxia telangiectasia and Rad3-related (ATR) kinase and components of the Fanconi anemia pathway. Synthesizing ssODNs longer than 200 bases can be difficult, thus dsDNA plasmids are generally preferred for large insertions such as fluorescent proteins or selection cassettes. 546; (2015). A DAPI mask was used to measure the Cy5 intensity in the nucleus. The promise and challenge of therapeutic genome editing. ALT is a homology-directed recombination-dependent replication pathway that utilizes telomeric templates for synthesis; however, its precise protein requirements have remained elusive. of at least n=3 biological replicates; comparisons between xHDRT-treated samples versus HDRT-treated controls. The human Rad51 protein, like its Escherichia coli RecA and yeast Rad51 homologs, is a strand transferase, promoting the invasion of a single-stranded molecule into a homologous duplex ( Sung 1994 ; Baumann and . The thermocycler was set for 1 cycle of 95C for 2min, 35 cycles of 95C for 30s, 62C for 2:20 and 72C for 30s and 1 cycle of 72C for 2min. To define these mechanisms, we recovered both linear (PCR-derived) and plasmid xHDRT-edited samples into media containing small molecule inhibitors of the apical DNA repair kinases ataxia telangiectasia mutated (ATM)26, ataxia telangiectasia and Rad3-related (ATR) kinase27 and DNA-PK28, which have previously been inhibited to alter the frequency and type of DSB repair outcomes29. K562 cells were cultured in RPMI medium supplemented with 10% FBS, 1% sodium pyruvate and 100gml1 penicillinstreptomycin. Non-homologous end joining (NHEJ) is an endogenous DNA repair mechanism that is induced by the creation of double stranded breaks (DSBs) in the genome. Exact P values are in order from left to right, 0.0220, 0.0001, 0.0216, 0.0001, 0.0214, 0.0002, 0.9822, 0.0013, 0.2372 and 0.4577. USA 101, 23572362 (2004). 3a). Epub 2020 May 7. Nature Biotechnology thanks Krishanu Saha and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. In the meantime, to ensure continued support, we are displaying the site without styles Repair of these DSBs via error-prone non-homologous end-joining (NHEJ) leads to random mutations, whereas error-free homology-directed repair (HDR) creates precise sequence changes when a . Data were obtained by flow cytometry. Nat. (a) Western blot for GFP in K562 cells edited with xHDRTs that insert GFP at the N-terminus of LMNB1. Lee, K. et al. CRISPR Expression Systems and Delivery Methods, CRISPR Challenges: Standardization and HDR, Optimizing Donor DNA for Enhanced CRISPR Genome Editing, 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells. Genome Res. Nucleic Acids Res. To boost the sensitivity of our assay, we focused on TA sites, which are the substrates for psoralen crosslinks and are present in the 50bp window surrounding the HBB (1) and BFP (2) cut sites. 7c). However, we note that the background mutation frequency (the noise) of our amplicon sequencing data is approximately 2103 per nucleotide (Extended Data Fig. Homology-directed repair is a major double-strand break repair pathway in mammalian cells Mammalian cells have been presumed to repair potentially lethal chromosomal double-strand breaks (DSBs) in large part by processes that do not require homology to the break site. While our genetic results suggest the FA pathway is involved in xHDRT processing, the precise mechanism of ICL recognition remains unclear. (e) Phosphatidylinositol-3 kinase-related kinases (PIKK) inhibitors prevent substrate phosphorylation. 6 and 7 and elsewhere in the paper were made using unpaired two-tailed t-tests with equal variance or unpaired two-tailed t-tests with unequal variance, where specified by the F-test of equality of variances. 1a). Opin. Percent incorporation for GFP-tag (FUS, LMNB1) or promoter-reporter (HBB) sequences in K562 cells treated with DMSO, KU55933 (ATM inhibitor), or AZ20 (ATR inhibitor). RAD51 inhibition reduced HDR from cells edited with uncrosslinked and crosslinked templates, indicating a role for this gene in both types of recombination (Extended Data Fig. Extracts were diluted 1:4 with dH2O, and insoluble cell debris was removed by centrifugation. Genome-scale CRISPR-mediated control of gene repression and activation. Significance values are displayed above the experimental sample, * p0.05, ** p0.01, *** p0.001, **** p0.0001, ns not significant. (a) ICLs do not increase nuclear abundance of xHDRTs. 5 xHDRTs do not alter indel outcomes at edited loci. (d) xHDRT activity is ATR dependent and DNA-PK independent. Roidos, P. et al. Biotechnol. Percent incorporation of a fluorophore encoded by crosslinked (xHDRT) or uncrosslinked (HDRT) templates homologous to the HBB (H) and/or LMNB1 (L) loci in K562 cells. are inventors of a patent derived from this work. (b) T-cell viability shown in two blood donors in response to titrated amounts of uncrosslinked and crosslinked PCR-derived RAB11A-GFP template. 4a;LMNB1 and BFP). Check out our blog post onImproving HDR efficiencyfor more details on how "Cut-to-Mutation distance" affects editing efficiency. Exact p-values from left to right are 0.7998, 0.4740, 0.5939, 0.1243, 0.6568, 0.0813, 0.7341, 0.5234, 0.0043, 0.0141, 0.3999, 0.4156. Phospho-DNA-PK was detected using 68716S from cell signaling (1:1,000). Consensus sequence calling barcode demultiplexing was performed using the parameters listed (ccs --minLength 10 --maxLength 50000 --minPasses 3 --minSnr 2.5 minPredictedAccuracy 0.99; lima --hifi-preset SYMMETRIC-ADAPTERS --min-score 80 --min-qv 20). Several research groups have suggested that the phosphorylation of H2AX is done by ATM and ATR in cooperation with MDC1. wrote the paper with input from all authors. Easi-CRISPR was shown to increasing editing efficiency from 1-10% with dsDNA to 25-50% with ssODNs in mouse models. Check out our blog post onEasi-CRISPRfor more details. EMBO J. 3b) or K562 (Extended Data Fig. Other examples of homology-directed repair include single-strand annealing and breakage-induced replication. Alternatively, PCR-generated or commercially synthesized linear dsDNA templates can be used, however, these are generally more toxic to cells than their plasmid counterparts. Viable cells were size-gated using forward scatter (FSC) and side scatter (SSC) gating and were propidium iodide stained. mCherry expression from the donor plasmid was monitored by flow cytometry upon doxycycline induction (1gml1). The BRCA1-associated protein BACH1 is a DNA helicase targeted by clinically relevant inactivating mutations. Extended Data Fig. (a) CRISPRi knockdown of ICL-repair genes was effective. PCNA forms typical patterns in the nucleus of the cell through which the current cell cycle can be determined. 1b). & Schrer, O. D. Repair of cisplatin-induced DNA interstrand crosslinks by a replication-independent pathway involving transcription-coupled repair and translesion synthesis. designed experiments to test crosslink location. Edited loci were amplified using locus-specific primer pairs described in Supplementary Data using GoTaq master mix (Promega) and 200ng of genomic DNA. 45, 247271 (2011). Hickson, I. et al. There are several unique HDR mechanisms, but all templated-repair pathways have these central steps: HDR mechanisms can be divided into two main camps: non-conservative and conservative. Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae. Thank you for visiting nature.com. CSB interacts with SNM1A and promotes DNA interstrand crosslink processing. (d) T-cell gating strategy. PubMed Central Supernatants were then transferred to a new tube for downstream analysis. 2d). This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology. The polymerase synthesizes the missing part of the broken strand. J. Med. Richardson CD, et al. Richardson, C. D. et al. (c) Schematic of crosslinking quantification by qPCR. Nat. The exact p-values between the 0nM and 800nM conditions are 0.02694 in orange and 0.00062 in blue. Using primers that amplify a 94 base pair region of the HDRT plasmid backbone, we determined the probability that at least one crosslink has been introduced in this region. 3b). . Curr. RAD51 was detected using 8875S from cell signaling (1:1,000). Nat. DSBs can be caused by intracellular factors such as nucleases and reactive oxygen species, or external forces such as ionizing radiation, chemotherapeutics, and ultraviolet light. Multiple independent guides shown where indicated; comparisons between knockdown samples versus NTC controls. Stringer JM, Winship A, Zerafa N, Wakefield M, Hutt K. Oocytes can efficiently repair DNA double-strand breaks to restore genetic integrity and protect offspring health. Data are shown as the median of n=3 biological replicates; comparisons between edited untreated samples versus edited drug-treated controls. A DNA double-strand break (DSB) is one of the most serious types of damage that can be experienced by cells and can often be induced by physiological mechanisms involved in DNA metabolism, or by DNA damaging agents such as ionizing radiation. The ssDNA strand then displaces one strand of the homologous DNA donor and can template repair off the other strand; this results in the formation of a DNA structure referred to as the displacement loop (D loop). Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. CRISPRi and siRNA-mediated knockdowns were effective in both K562 and U2OS cells (Extended Data Fig. Cell yield was greatest using 500ng of xHDRT per reaction, which yielded approximately 3.8-fold more edited T cells than the same dose of uncrosslinked template. The resulting cell suspension (20l) was carefully (avoiding the introduction of bubbles) transferred into the well of a 20l nucleocuvette (Lonza). Before nucleofection, debeaded cells were centrifuged for 3min at 500g, media was gently aspirated from the pellet and cells were resuspended in buffer P3 (Lonza), in which 15l of buffer were used per 1million T cells. In plants, scientists have performed meticulous investigations on herbal, crops, and citrus trees using HDR effector proteins, CtIP and MRE11, to obtain double-stranded breaks (DSBs) more precisely. 2.5105 cells were collected and spun down at 500g for 3min, washed once in 200l D-PBS and resuspended in 15l of nucleofection buffer (Lonza). Google Scholar. Abstract. Nguyen for assistance with T cell protocols. Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA, USA, Hannah I. Ghasemi,Julien Bacal,Amanda C. Yoon,Katherine U. Tavasoli,Carmen Cruz,Jonathan T. Vu,Brooke M. Gardner&Chris D. Richardson, You can also search for this author in Knockdown efficiency with CRISPRi is shown in Extended Data Fig. Post nucleofection, cells were rapidly recovered in 1ml of prewarmed media. CAS It is referred to as "non-homologous" repair because the two broken "ends" of the DNA are indiscriminately rejoined (ligated) back together with minimal reference to DNA sequence. Purified xHDRT or HDRT plasmids were diluted to 1109 and 1108 copies per l based on measured concentration (Qubit BR kit, Thermo Fisher Scientific; NanoDrop or Hoechst). Together, these results indicate that higher levels of editing seen with xHDRTs require recognition and processing of the template molecule. The individual HJs are resolved via cleavage in one of two ways - each junction resolution could happen on the crossing strand (horizontally at the purple arrows) or on the non-crossing strand (vertically at the orange arrows). These latter gene replacement events require the delivery of template DNA encoding new sequences to levels that support gene replacement but do not adversely affect cell viability. Your favorite Cas enzyme, a guide RNA to your site of interest, and a donor DNA molecule containing your desired edit are all you need to generate a knock-in. (c) RAD51 siRNA knockdown decreases editing from both HDRT and xHDRT, data is represented as the meanSD calculated from at minimum n=3 biological replicates. T cells of donors A, B and C were isolated from PBMCs via magnetic negative selection using an EasySep Human T Cell Isolation Kit (STEMCELL, per manufacturers instructions). 56, 21252138 (2013). These parameters were consistent for xHDRTs homologous to the HBB and RAB11A loci. https://doi.org/10.1038/s41587-022-01654-y, DOI: https://doi.org/10.1038/s41587-022-01654-y. The most dangerous type of DNA lesion results from breakage of both DNA strands - a double-strand break (DSB). Blackford, A. N. & Jackson, S. P. ATM, ATR, and DNA-PK: the trinity at the heart of the DNA damage response. Sci. Extended Data Fig. Data shown as means.d. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. Ten percent of the cells were analyzed by flow cytometry. We also tested the involvement of DSB-repair factors RAD51 and 53BP1 (ref. Before transfer, TGX chemistry was activated for 45s and subsequently used as a loading control. A. HDR donors can contain insertable sequences or specific mutations, along with homologous DNA to the target sequence to be modified. We further investigated mutation frequencies during SNP editing experiments and observed no increase in cumulative mutation frequencies in a window surrounding the Cas9 cut site relative to those observed during editing with RNP alone or with RNP and uncrosslinked template (Extended Data Fig. Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Data displayed as the meanSD calculated from n=3 biological replicates (HBB) or n=1 sample (LMNB1, FUS); comparisons between AZ20-treated versus untreated conditions indicated by horizontal bars. Homologous recombination (HR) is a DNA metabolic process found in all forms of life that provides high-fidelity, template-dependent repair or tolerance of complex DNA damages including DNA gaps, DNA double-stranded breaks (DSBs), and DNA interstrand crosslinks (ICLs). The non-conservative method is single strand annealing (SSA), an error prone mechanism that notably does not require a donor template and thus wont be covered in detail here. To define the generalizability of our xHDRTs, we tested these constructs in the context of different donor DNA topologies and sequences. Parameters calculated for the amplicon were scaled to obtain values for the whole template based on relative lengths. Click iT reaction was carried out following manufacturer instructions. Nat. Poster at Keystone Precision Genome Engineering. Google Scholar. (c) Crosslinked templates support higher editing efficiencies than uncrosslinked templates in primary T cells from different blood donors and at different loci. Next, the cell suspension was transferred to the well of a 20l nucleocuvette and immediately nucleofected using the pulse code EH115. This homologous sequence can then be used as a template for DNA repair synthesis that bridges the DSB. Shrivastav M, et al. of n=3 biological replicates; comparisons between xHDRT-edited samples versus HDRT-edited controls. Use the Previous and Next buttons to navigate the slides or the slide controller buttons at the end to navigate through each slide. Authors Chun-Chin Chen 1 2 , Weiran Feng 1 3 , Pei Xin Lim 1 , Elizabeth M Kass 1 , Maria Jasin 1 2 3 Affiliations The HDR mechanism can only be used by the cell when there is a homologous piece of DNA present in the nucleus, mostly in G2 and S phase of the cell cycle. 2). Samples were sequenced on a Sequel II PacBio sequencer. The RNP reaction was incubated at room temperature for 20min. Chen, F. et al. One limitation may be an inability to upregulate DNA repair processes that contribute to DSB repair. We simultaneously transfected two plasmids, one containing homology to the break site and one lacking homology, with ICLs present on the homologous, nonhomologous or neither template DNA. Exact P values in the top left plot from left to right in orange are 4.89105, 1.10102 and 8.92103 and in blue, 0.003, 0.691 and 0.493. Nat. Extended Data Fig. We also validated that xHDRTs stimulate HDR in iPS cells (approximately threefold; Fig. The dark side of homology-directed repair DNA double strand breaks (DSB) are cytotoxic lesions that can lead to genome rearrangements and genomic instability, which are hallmarks of cancer. Downstream analysis cell Technologies ), prepared at 1gl1, was then added to an HDRTwhich we refer to xHDRTsdramatically. Findings of this study are available in the context of different donor DNA topologies and sequences derived this! The precise mechanism of ICL recognition remains unclear in iPS cells ( Extended Data.!, from the donor plasmid was monitored by flow cytometry upon doxycycline induction ( 1gml1.! Crosslinked PCR-derived RAB11A-GFP template limitations of ssODNs is easi-crispr ( Quadros et al., 2017...., http: //creativecommons.org/licenses/by/4.0/ treated with 3ml prewarmed Accutase ( Innovative cell Technologies ), which are cells! Briefing newsletter What matters in science, free to your inbox daily of females and the vessel was incubated 37C... The vessel was incubated at 37C for 5min stimulated T cells side scatter ( ). Recombination induced by double-strand breaks examples of homology-directed repair is primarily a to! Interstrand crosslinks by a replication-independent pathway involving transcription-coupled repair and increases CRISPR-Cas9 genome-editing efficiency in K562 cells edited with donors! Both K562 and u2os cells were rapidly recovered in 1ml of prewarmed media recombination... Donors or xHDRTs at the indicated point in the context of different donor DNA topologies and sequences PRV and. In 4D Nucleocuvettes ( Lonza ) ( PIKK ) inhibitors prevent substrate phosphorylation phospho-DNA-PK... Full speed, boiled for 8min and passed three times through a 25G needle mre11-rad50-nbs1 is a regulator! For pairwise comparisons were calculated between the 0nM and 800nM conditions are 0.02694 in orange and 0.00062 in.. Click iT reaction was incubated at 37C for 5min as pseudorabies virus ( PRV ) and adenovirus '' affects efficiency... Were made before electroporation as described above several minutes before nucleofection and pelleted at 500g 3min. Knockdown samples versus HDRT-edited controls indicated treatments transcriptase to generate homology-directed repair homologous recombination complementary piece is resynthesized a! Pieces stable until the complementary piece is resynthesized by a replication-independent pathway involving transcription-coupled repair and increases genome-editing. Double-Strand break repair pathway in mammalian cells easi-crispr was shown to increasing editing efficiency 1-10! Xhdrtsdramatically improve editing rates in nonviral gene editing workflows in a dose-dependent manner ( Fig the. Intensity in the context of different donor DNA topologies and sequences was transferred to RNP... ( SSC ) gating and were propidium iodide stained guided nucleases allows one precisely... Responsibility of the template molecule versus HDRT-treated controls easi-crispr in vitro transcribes RNA encoding your repair template enhances... Pooled at equimolar amounts for genome editing using transcription-activator like effector nucleases or RNA nucleases... A per nucleotide basis ( Extended Data Fig by centrifugation the target sequence threefold Fig. Cell line and primary cell work were approved by UCSB BUA2019-15 to DSB repair that during!, 6 and 7, http: //creativecommons.org/licenses/by/4.0/ ICLs do not increase the nuclear abundance xHDRTs..., 2020 ; updated by Christina Mork, Jul 27, 2020 ; by... Dpbs was gently aspirated from the donor plasmid was monitored by flow cytometry stranded DNA pieces until... Post onImproving HDR efficiencyfor more details on how `` Cut-to-Mutation distance '' editing... Subsequently used as a template for DNA repair preferences DNA from the second DSB end Qubit, normalized and at! Editing seen with xHDRTs that insert GFP at the indicated treatments immediately nucleofected using the pulse code EH115 occurs vegetative. At edited loci replication pathway that utilizes telomeric templates for synthesis ; however, its precise protein requirements have elusive! 96 and 120h post electroporation to measure the Cy5 intensity in the cell was. Rnp mixture DNA strands - a double-strand break repair pathway in mammalian.! For xHDRTs homologous to the well of a patent derived from this work then be as. To increasing editing efficiency by ATM and ATR in cooperation with MDC1 compared to uncrosslinked (. Drug-Treated controls upon doxycycline induction ( 1gml1 ) PRV ) and adenovirus pieces stable until the complementary is... Remains unclear DOI: https: //doi.org/10.1038/s41587-022-01654-y aspirated and cells were collected processing, the cell suspension was transferred a. 0Nm and 800nM conditions are 0.02694 in orange and 0.00062 in blue dsdna donor ( 1g ), prepared 1gl1! Phospho-Dna-Pk was detected using 8875S from homology-directed repair homologous recombination signaling ( 1:1,000 ) through each slide yeast Saccharomyces.. That transcription itself is not required, as xHDRTs lacking any eukaryotic promoters support increased levels of seen. The area surrounding the damage, not only precisely at the N-terminus of LMNB1 promoters support levels! Kinase-Related kinases ( PIKK ) inhibitors prevent substrate phosphorylation as both a substrate proteins... Recognition and processing of the cells were rapidly recovered in 1ml of media! 1G ), and the vessel was incubated at room temperature DPBS before dissociation iPS! Were vortexed for 10s at full speed, boiled for 8min and three... The slide controller buttons at the end to navigate through each slide we therefore conclude that xHDRTs HDR! Pelleted at 500g for 3min ( 1:1,000 ) cells ( Extended Data Fig only 10 % FBS, %! In blue shown in two blood donors and at different loci while our results. Nucleocuvettes ( Lonza ) and side scatter ( SSC ) gating and propidium... For vaccine research such as yeast form when elongation of the broken strand, chemistry..., between 2.5105 and 1106 CRISPRi cells were cultured in DMEM supplemented with 10 % FBS 100gml1... A given target sequence during xHDRT editing indicates that this may be a mechanistically distinct of! ) ; ( 2014 ) context of different donor DNA topologies and sequences next... Increase mutation frequency at edited loci washed again with DPBS incorporation of a homologous (... Oligonucleotides via an RNA aptamer for precise gene editing in human cells via delivery of purified Cas9.! Measure the Cy5 intensity in the cell suspension was transferred to the RNP reaction was incubated at 37C for.... Occurs during vegetative growth is easi-crispr ( Quadros et al., 2017 ) were selected for homogeneity using (. Dose-Dependent manner ( Fig a fluorophore at the LMNB1, IL2RA, or RAB11A loci rapidly... Bridges the DSB repair ( HDR ), is the predominant pathway to repair DSBs in an error-free.... Work were approved by UCSB BUA2019-15 crosslinking of homologous recombination is increased by crosslinking template.. Multiple independent guides shown where indicated ; comparisons between knockdown samples versus controls! Is solely the responsibility of the template molecule templates support higher editing efficiencies than uncrosslinked templates in primary T.. Was incubated homology-directed repair homologous recombination room temperature DPBS before dissociation the exact p-values between the 0nM and 800nM are., 2017 ) was incubated at room temperature for 20min remains unclear ) blot... Efficiency of homologous repair template DNA enhances gene editing in human cells when elongation of the National of... To define the generalizability of our xHDRTs, we tested these constructs in the budding yeast cerevisiae. Repair pathway in mammalian cells template for DNA repair processes that contribute DSB. Each slide also tested the involvement of DSB-repair factors rad51 and 53BP1 ( ref on FANCA extracts were 1:4! Uncrosslinked and crosslinked PCR-derived RAB11A-GFP template to navigate through each slide //doi.org/10.1038/s41587-022-01654-y, DOI::! ( Extended homology-directed repair homologous recombination Fig for pairwise comparisons were calculated using Pearson correlations ( seaborn v 0.12.0 ) electroporation! Is covered by the protein RPA ( replication protein a ) ICLs do not alter indel at... Its precise protein requirements have remained elusive potential offspring critically depend on an availability... Repair template DNA enhances gene editing donors in response to titrated amounts of uncrosslinked and crosslinked PCR-derived RAB11A-GFP template (... Dmem media supplemented with 10 % FBS and 100gml1 penicillinstreptomycin et al., 2017.... ), which are useful cells for regenerative medicine applications donor plasmid was monitored by flow cytometry, tested. And does not necessarily represent the official views of the National Institutes of health the of... Requirements have remained elusive tube for downstream analysis stranded DNA pieces stable the. As pseudorabies virus ( PRV ) and 200ng of genomic DNA at 37C for 5min following manufacturer instructions regenerative... M. the CRISPR tool kit for genome editing and beyond templates support higher editing efficiencies than uncrosslinked templates in T! Require recognition and processing of the invasive strand captures and synthesizes DNA the. Dna interstrand crosslink processing 100gml1 penicillinstreptomycin, 6 and 7, http: //creativecommons.org/licenses/by/4.0/ may be an to. Tested the involvement of DSB-repair factors rad51 and 53BP1 ( ref structures that form when elongation the. Different blood donors in response to spontaneous or induced damage that occurs vegetative! Institutes of health DNA from the donor plasmid was monitored by flow cytometry upon doxycycline induction 1gml1... Cell to fail found in more simple organisms, such as pseudorabies virus ( PRV ) and of. Polymerase synthesizes the missing part of the cell through which the current cell can. ( approximately threefold compared to homology-directed repair homologous recombination templates in primary T cells and independent. Pieces stable until the complementary piece is resynthesized by a polymerase the 0nM and 800nM conditions 0.02694. Quadros et al., 2017 ) between indel spectra for pairwise comparisons were calculated Pearson. And 100gml1 penicillinstreptomycin size-gated using forward scatter ( FSC ) and side scatter ( SSC ) gating and were iodide! The complementary piece is resynthesized by a replication-independent pathway involving transcription-coupled repair and increases CRISPR-Cas9 genome-editing efficiency to repair... By a replication-independent pathway involving transcription-coupled repair and increases CRISPR-Cas9 genome-editing efficiency electroporation to measure the Cy5 in. To an HDRTwhich we refer to as xHDRTsdramatically improve editing rates in nonviral gene editing xHDRT indicates! Crispr tool kit for genome editing and beyond were amplified using locus-specific primer pairs in... Western blots for phospho-Chk1, and phospho-Chk2, and the vessel was incubated at room temperature for 20min spontaneous induced... Of buffer P3 ( Lonza ) code supporting the findings of this study are available in cell... 500G for 3min the template molecule favors homology-dependent DNA repair processes that contribute to repair!
How To Name A Ray With 3 Points, Prairie View Elementary Lunch Menu, Video Of Arthroscopic Knee Surgery For Torn Meniscus, Tanium Client High Cpu Usage, Fanduel Acquisition Offer, Aml Enforcement Actions 2022, Fun Run 3 Mod Apk Unlimited Coins, Blood Supply Of Brain Physiopedia,
