Single-strand Annealing Repair Research Articles

Nonhomologous tails direct heteroduplex rejection and mismatch correction during single-strand annealing in Saccharomyces cerevisiae.

Single-strand annealing (SSA) is initiated when a double strand break (DSB) occurs between two flanking repeated sequences, resulting in a deletion that leaves a single copy of the repeat. We studied budding yeast strains carrying two 200-bp URA3 sequences separated by 2.6 kb of spacer DNA (phage lambda) in which a site-specific DSB can be created by HO or Cas9 endonucleases. Repeat-mediated deletion requires removal of long 3'-ended single-stranded tails (flaps) by Rad1-Rad10 with the assistance of Msh2-Msh3, Saw1 and Slx4. A natural 3% divergence of unequally spaced heterologies between these repeats (designated F and A) causes a significant reduction in the frequency of SSA repair. This decrease is caused by heteroduplex rejection in which mismatches (MMs) in the annealed intermediate are recognized by the MutS (Msh2 and Msh6) components of the MM repair (MMR) pathway coupled to unwinding of the duplex by the Sgs1-Rmi1-Top3 helicase. MutL homologs, Mlh1-Pms1 (MutL), are not required for rejection but play their expected role in mismatch correction. Remarkably, heteroduplex rejection is very low in strains where the divergent repeats were immediately adjacent (Tailless strains) and the DSB was induced by Cas9. These results suggest that the presence of nonhomologous tails strongly stimulates heteroduplex rejection in SSA. DNA sequencing analysis of SSA products from the FA Tailed strain showed a gradient of correction favoring the sequence opposite each 3' end of the annealed strand. Mismatches located in the center of the repair intermediate were corrected by Msh2-Msh6 mediated mismatch correction, while correction of MMs at the extremity of the SSA intermediate often appears to use a different mechanism, possibly by 3' nonhomologous tail removal that includes part of the homologous sequence. In contrast, in FA Tailless strains there was a uniform repair of the MMs across the repeat. A distinctive pattern of correction was found in the absence of MSH2, in both Tailed and Tailless strains, different from the spectrum seen in a msh3Δ msh6Δ double mutant. Previous work has shown that SSA is Rad51-independent but dependent on the strand annealing activity of Rad52. However Rad52 becomes dispensable in a Tailless construct where the DSB is induced by Cas9 or in transformation of a plasmid where SSA occurs in the absence of nonhomologous tails.

A dual-function selection system enables positive selection of multigene CRISPR mutants and negative selection of Cas9-free progeny in Arabidopsis

The CRISPR/Cas9 technology revolutionizes targeted gene knockout in diverse organisms including plants. However, screening edited alleles, particularly those with multiplex editing, from herbicide- or antibiotic-resistant transgenic plants and segregating out the Cas9 transgene represent two laborious processes. Current solutions to facilitate these processes rely on different selection markers. Here, by taking advantage of the opposite functions of a d-amino acid oxidase (DAO) in detoxifying d-serine and in metabolizing non-toxic d-valine to a cytotoxic product, we develop a DAO-based selection system that simultaneously enables the enrichment of multigene edited alleles and elimination of Cas9-containing progeny in Arabidopsis thaliana. Among five DAOs tested in Escherichia coli, the one encoded by Trigonopsis variabilis (TvDAO) could confer slightly stronger d-serine resistance than other homologs. Transgenic expression of TvDAO in Arabidopsis allowed a clear distinction between transgenic and non-transgenic plants in both d-serine-conditioned positive selection and d-valine-conditioned negative selection. As a proof of concept, we combined CRISPR-induced single-strand annealing repair of a dead TvDAO with d-serine-based positive selection to help identify transgenic plants with multiplex editing, where d-serine-resistant plants exhibited considerably higher co-editing frequencies at three endogenous target genes than those selected by hygromycin. Subsequently, d-valine-based negative selection successfully removed Cas9 and TvDAO transgenes from the survival offspring carrying inherited mutations. Collectively, this work provides a novel strategy to ease CRISPR mutant identification and Cas9 transgene elimination using a single selection marker, which promises more efficient and simplified multiplex CRISPR editing in plants.

Noodles, the all-in-one system for on-target efficiency analysis of CRISPR guide RNAs

The efficiency of clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA (gRNA) targeting is critical for CRISPR associated protein 9 (Cas9)-dependent genomic modifications. Here, we developed Noodles, an all-in-one system to test the on-target activity of gRNAs easily and efficiently. Single-strand annealing repair mechanism of the split luciferase gene allows a positive selection of gRNAs efficiently driving nuclease activity of Cas9 from Streptococcus pyogenes (SpCas9). Our system can reliably validate in silico-predicted gRNAs before implementing them for in vitro and in vivo applications. Altogether, Noodles might be an asset for researchers and bioengineers, saving their time and efforts, while keeping the screening efficient and sensitive.•All-in-one dual-luciferase system to easily probe on-target activity of gRNAs based on homology-directed repair mechanism.•Easy-to-subclone spCas9-based 2-plasmid system comprising Renilla luciferase for transfection efficiency control.

Fanconi anemia associated protein 20 (FAAP20) plays an essential role in homology-directed repair of DNA double-strand breaks

FAAP20 is a Fanconi anemia (FA) protein that associates with the FA core complex to promote FANCD2/FANCI monoubiquitination and activate the damage response to interstrand crosslink damage. Here, we report that FAAP20 has a marked role in homologous recombination at a DNA double-strand break not associated with an ICL and separable from its binding partner FANCA. While FAAP20’s role in homologous recombination is not dependent on FANCA, we found that FAAP20 stimulates FANCA’s biochemical activity in vitro and participates in the single-strand annealing pathway of double-strand break repair in a FANCA-dependent manner. This indicates that FAAP20 has roles in several homology-directed repair pathways. Like other homology-directed repair factors, FAAP20 loss causes a reduction in nuclear RAD51 Irradiation-induced foci; and sensitizes cancer cells to ionizing radiation and PARP inhibition. In summary, FAAP20 participates in DNA double strand break repair by supporting homologous recombination in a non-redundant manner to FANCA, and single-strand annealing repair via FANCA-mediated strand annealing activity.

An ELISA-based platform for rapid identification of structure-dependent nucleic acid–protein interactions detects novel DNA triplex interactors

Unusual nucleic acid structures play vital roles as intermediates in many cellular processes and, in the case of peptide nucleic acid (PNA)–mediated triplexes, are leveraged as tools for therapeutic gene editing. However, due to their transient nature, an understanding of the factors that interact with and process dynamic nucleic acid structures remains limited. Here, we developed snapELISA (structure-specific nucleic acid-binding protein ELISA), a rapid high-throughput platform to interrogate and compare up to 2688 parallel nucleic acid structure–protein interactions in vitro. We applied this system to both triplex-forming oligonucleotide–induced DNA triplexes and DNA-bound PNA heterotriplexes to describe the identification of previously known and novel interactors for both structures. For PNA heterotriplex recognition analyses, snapELISA identified factors implicated in nucleotide excision repair (XPA, XPC), single-strand annealing repair (RAD52), and recombination intermediate structure binding (TOP3A, BLM, MUS81). We went on to validate selected factor localization to genome-targeted PNA structures within clinically relevant loci in human cells. Surprisingly, these results demonstrated XRCC5 localization to PNA triplex-forming sites in the genome, suggesting the presence of a double-strand break intermediate. These results describe a powerful comparative approach for identifying structure-specific nucleic acid interactions and expand our understanding of the mechanisms of triplex structure recognition and repair.

A Simulated Shift Work Schedule Does Not Increase DNA Double-Strand Break Repair by NHEJ in the Drosophila Rr3 System

Long-term shift work is widely believed to increase the risk of certain cancers, but conflicting findings between studies render this association unclear. Evidence of interplay between the circadian clock, cell cycle regulation, and DNA damage detection machinery suggests the possibility that circadian rhythm disruption consequent to shift work could alter the DNA double-strand break (DSB) repair pathway usage to favor mutagenic non-homologous end-joining (NHEJ) repair. To test this hypothesis, we compared relative usage of NHEJ and single-strand annealing (SSA) repair of a complementary ended chromosomal double-stranded break using the Repair Reporter 3 (Rr3) system in Drosophila between flies reared on 12:12 and 8:8 (simulated shift work) light:dark schedules. Actimetric analysis showed that the 8:8 light:dark schedule effectively disrupted the rhythms in locomotor output. Inaccurate NHEJ repair was not a frequent outcome in this system overall, and no significant difference was seen in the usage of NHEJ or SSA repair between the control and simulated shift work schedules. We conclude that this circadian disruption regimen does not alter the usage of mutagenic NHEJ DSB repair in the Drosophila male pre-meiotic germline, in the context of the Rr3 system.

SAW1 is increasingly required to recruit Rad10 as SSA flap-length increases from 20 to 50 bases in single-strand annealing in S. cerevisiae

SAW1 is required by the Rad1-Rad10 nuclease for efficient removal of 3′ non-homologous DNA ends (flaps) formed as intermediates during two modes of double-strand break repair in S. cerevisiae, single-strand annealing (SSA) and synthesis-dependent strand annealing (SDSA). Saw1 was shown in vitro to exhibit increasing affinity for flap DNAs as flap lengths varied from 0 to 40 deoxynucleotides (nt) with almost no binding observed when flaps were shorter than 10 nt. Accordingly, our prior in vivo fluorescence microscopy investigation showed that SAW1 was not required for recruitment of Rad10-YFP to DNA double-strand breaks (DSBs) when flaps were ∼10 nt, but it was required when flaps were ∼500 nt in G1 phase of the cell cycle. We were curious whether we would also observe an increased requirement of SAW1 for Rad10 recruitment in vivo as flaps varied from ∼20 to 50 nt, as was shown in vitro. In this investigation, we utilized SSA substrates that generate 20, 30, and 50 nt flaps in vivo in fluorescence microscopy assays and determined that SAW1 becomes increasingly necessary for SSA starting at about ∼20 nt and is completely required at ∼50 nt. Quantitative PCR experiments corroborate these results by demonstrating that repair product formation decreases in the absence of SAW1 as flap length increases. Experiments with strains containing fluorescently labeled Saw1 (Saw1-CFP) show that Saw1 localizes with Rad10 at SSA foci and that about half of the foci containing Rad10 at DSBs do not contain Saw1. Colocalization patterns of Saw1-CFP are consistent regardless of the flap length of the substrate and are roughly similar in all phases of the cell cycle. Together, these data show that Saw1 becomes increasingly important for Rad1-Rad10 recruitment and SSA repair in the ∼20–50 nt flap range, and Saw1 is present at repair sites even when not required and may depart the repair site ahead of Rad1-Rad10.

The Role of Drosophila CtIP in Homology-Directed Repair of DNA Double-Strand Breaks.

DNA double-strand breaks (DSBs) are a particularly genotoxic type of DNA damage that can result in chromosomal aberrations. Thus, proper repair of DSBs is essential to maintaining genome integrity. DSBs can be repaired by non-homologous end joining (NHEJ), where ends are processed before joining through ligation. Alternatively, DSBs can be repaired through homology-directed repair, either by homologous recombination (HR) or single-strand annealing (SSA). Both types of homology-directed repair are initiated by DNA end resection. In cultured human cells, the protein CtIP has been shown to play a role in DNA end resection through its interactions with CDK, BRCA1, DNA2, and the MRN complex. To elucidate the role of CtIP in a multicellular context, CRISPR/Cas9 genome editing was used to create a DmCtIPΔ allele in Drosophila melanogaster. Using the DSB repair reporter assay direct repeat of white (DR-white), a two-fold decrease in HR in DmCtIPΔ/Δ mutants was observed when compared to heterozygous controls. However, analysis of HR gene conversion tracts (GCTs) suggests DmCtIP plays a minimal role in determining GCT length. To assess the function of DmCtIP on both short (~550 bp) and long (~3.6 kb) end resection, modified homology-directed SSA repair assays were implemented, resulting in a two-fold decrease in SSA repair in both short and extensive end resection requirements in the DmCtIPΔ/Δ mutants compared to heterozygote controls. Through these analyses, we affirmed the importance of end resection on DSB repair pathway choice in multicellular systems, described the function of DmCtIP in short and extensive DNA end resection, and determined the impact of end resection on GCT length during HR.

Analysis of DNA Double-Strand Break End Resection and Single-Strand Annealing in S. pombe.

DNA double-strand break (DSB) end resection is an essential step for homologous recombination. It generates 3' single-stranded DNA needed for the loading of the strand exchange proteins and DNA damage checkpoint proteins. To study the mechanism of end resection in fission yeast, we apply a robust, quantitative and inducible assay. Resection is followed at a single per genome DSB synchronously generated by the tet-inducible I-PpoI endonuclease. An additional assay to follow resection involves recombination between two direct repeats by single-strand annealing (SSA), since SSA requires extensive resection to expose two single-strand repeats for annealing. The kinetics of resection and SSA repair are then measured using Southern blots.

RNF8 has both KU-dependent and independent roles in chromosomal break repair.

Chromosomal double strand breaks (DSBs) can initiate several signaling events, such as ubiquitination, however the precise influence of such signaling on DSB repair outcomes remains poorly understood. With an RNA interference screen, we found that the E3 ubiquitin ligase RNF8 suppresses a deletion rearrangement mediated by canonical non-homologous end joining (C-NHEJ). We also found that RNF8 suppresses EJ without insertion/deletion mutations, which is a hallmark of C-NHEJ. Conversely, RNF8 promotes alternative EJ (ALT-EJ) events involving microhomology that is embedded from the edge of the DSB. These ALT-EJ events likely require limited end resection, whereas RNF8 is not required for single-strand annealing repair involving extensive end resection. Thus, RNF8 appears to specifically facilitate repair events requiring limited end resection, which we find is dependent on the DSB end protection factor KU. However, we also find that RNF8 is important for homology-directed repair (HDR) independently of KU, which appears linked to promoting PALB2 function. Finally, the influence of RNF8 on EJ is distinct from 53BP1 and the ALT-EJ factor, POLQ. We suggest that RNF8 mediates both ALT-EJ and HDR, but via distinct mechanisms, since only the former is dependent on KU.

The effects of varying DNA flap length from 20 to 50 deoxynucleotides on the mechanism of single‐strand annealing in Saccharomyces cerevisiae

Mutagens constantly threaten the integrity of genetic material by causing damage in the form of double‐strand breaks (DSBs). Such damage is typically repaired through various modes of homologous recombination (HR). This project focuses on a form of HR known as single‐strand annealing (SSA), which is triggered when a DSB is located between two DNA repeats. This pathway generates 3' overhanging single‐stranded DNA “flaps” which are cleaved by the Rad1‐Rad10 endonuclease, aided by the mediator protein Saw1 in S. cerevisiae. Recent literature has demonstrated that Saw1‐dependent recruitment of Rad1‐Rad10 to damage sites, and SSA repair proficiency, are both a function of flap length. In this study, we set out to elucidate the precise flap length threshold requiring Saw1 for optimal SSA repair in vivo through fluorescence microscopy and real‐time quantitative PCR (qPCR).In order to investigate the importance of DNA flap length on Rad1‐Rad10 and Saw1 recruitment, we designed yeast strains generating 20, 30, or 50 nt flaps during SSA. A DSB was induced at a fluorescently labeled site in the yeast genome (DSB‐RFP), allowing us to monitor the recruitment of Rad10‐YFP via fluorescence microscopy. The percentages of cells containing colocalized foci in strains either wild‐type or mutant in Saw1 were quantified, revealing that Saw1‐mediated recruitment of the Rad1‐Rad10 complex increased with flap length. Surprisingly, we found that Saw1 was actively recruited to the DSB site across all flap length substrates via experiments with a Saw1‐CFP strain. Analyses via qPCR were also carried out to quantify SSA repair products formed in WT and saw1Δstrains. The data show diminished repair product formation in the absence of Saw1 as flap lengths increased. Together, these results suggest that Saw1 is required for Rad1‐Rad10 recruitment and efficient SSA repair in longer flap substrates with ~20–30 nt representing the key size triggering Saw1‐dependence for repair. Current efforts are underway to determine the Saw1‐dependence of proficient SSA repair under conditions in which background induction is not amplified by qPCR.Support or Funding InformationThe authors thank our Department and the NIH SC3 program for funding.

The Role of SGS1 Helicase in Mismatch Repair During Single Strand Annealing

In humans, interspersed repeated sequences constitute at least 35% of the genome. Single Strand Annealing (SSA) is a repair pathway which occurs when a double strand break is produced between homologous repeats. The product of SSA is a single copy of one of the homologous repeats. As such SSA is a highly mutagenic repair pathway. If an SSA event occurs and consequently deletes important genes such as BRAC2, which itself contains repeats, the result can be catastrophic.To study mismatch correction (MMC) during SSA, a yeast strain was created to have 7 mismatches (6 base‐pair substitutions and 1‐bp insertion/deletion), resulting in a 3% divergence between the repeats. Previous results show that strains with 3% divergence between the repeats, designated as the F‐A strain, reduced SSA two‐fold compared to the identical strains, designated as the A‐A strain. This reduction in SSA is due to heteroduplex rejection which occurs when the mismatch repair complex, MSH2‐MSH6, recognizes the mismatches in the DNA‐duplex and stimulates the unwinding of the DNA via SGS1 helicase, resulting in cell death. In the absence of heteroduplex, MSH2‐MSH3 will bind branched SSA intermediate, and recruit RAD1‐RAD10 to remove the non‐homologous tails.Sgs1Δ results in a very efficient SSA repair, with 2 folds increased viability, in both F‐A and A‐A strains, compared to WT strains as SGS1 is required for heteroduplex rejection. Sequencing analysis of sgs1Δ F‐A strain shows an asymmetric gradient of correction favoring the F fragment, similar to the WT strain, suggesting that SGS1 has minimal effects on MMC outcomes. In both sgs1Δ and WT strains tailless strains, there is a uniform correction of MMs, suggesting that the non‐homologous tails influence MMC. All together current data suggests that MMC is more likely to occur during replication, once the SSA repair is completed. Future directions include knocking‐out MLH1, a mismatch repair protein shown to interact with SGS1, and investigating if sectored colonies, cells with unrepaired mismatches is due to replication.Support or Funding InformationBrandeis Division of Science Summer Research Fellowship

Chasing the backup pathway for therapeutic opportunities in cancer

BRCA mutations are usually associated with the most aggressive subtypes of cancers such as triple‐negative breast cancer and high‐grade serous ovarian cancer. Finding a drug that can address the unique molecular features in these subtypes of cancer continues to be the best hope for treatment. In tumors with impaired BRCA‐pathway, RAD52 is a candidate for targeted inhibition as it is known to take a leading role in homologous recombination repair (HRR) of DNA double‐strand breaks (DSBs) in BRCA‐deficient cancer cells [1]. Additionally, RAD52 is the main player in single‐strand annealing (SSA), which has been reported to increase activity in a number of cancers. When RAD52 joins the DSB repair complex, RPA has already bound ssDNA and the resulting protein‐protein interaction (PPI) becomes instrumental for repair to proceed. Past work showed that conformational changes offered by RPA interaction modulate RAD52 activity [2]. A combined understanding of these bimolecular studies and the value of RAD52 in tumorigenesis were motivating factors for the development of a Fluorescence‐based protein‐protein Interaction Assay (FluorIA) [3]. FluorIA provided a tool to assess domains on RPA and RAD52 important for the PPI (Figure 1). The binding affinities and thermodynamics of those interacting domains were then evaluated by methods such as surface plasmon resonance and isothermal titration calorimetry to characterize RPA:RAD52 interaction as a vital therapeutic target.FluorIA was also used as a high throughput assay to screen over 100,000 potential small molecule inhibitors (SMIs). Eleven hits were identified, three of which are FDA approved drugs (Fig. 2). Characterization experiments thus far suggest a potential impact for at least one of these three drugs on SSA repair of DSBs. Our findings may offer molecular insights into a new mechanism of action for these drugs given their reported varied sensitivity in patients carrying BRCA deficiencies. The ultimate goal is to therapeutically target the addiction of cancer cells for DNA repair. However, we anticipate that a broader understanding of the mechanisms of action for the existing cancer drugs we screened would support the effort towards personalized medicine.Support or Funding Information1) SBMB Fellowship from U.S. Department of Education GAANN (P200A120231) 2) NASA Nebraska Space Grant: Graduate fellowship 3) Nebraska Department of Health and Human services (DHHS) LB506 4) Nebraska Research Initiative 5) Fred & Pamela Buffett Cancer Center Support Grant (P30CA036727) pilot project fundingCompetitive binding in FluorIA. A) Domain map of RAD52. B) SDS‐PAGE gel shown full‐length RAD52(1‐418) to the right of the molecular weight marker, RAD52(1‐303), and RAD52(1‐212). C) FluorIA reaction in which 20 pmol of EGFP tagged full‐length RAD52 is incubated with RPA alone or with a 100 pmol of unlabeled RAD52(1‐303) or (1‐212) to compete with EGFP‐RAD52 for RPA binding. Reduction in RFU signal signifies inhibition of RPA:RAD52 complex formation by the unlabeled RAD52 construct.Figure 1Inhibiti of RPA:RAD52 complex by the SMI leads. Hits are designated with the letters P or C according to the first letter of the source library name. The best IC50 are P1, 110 μM; P2, 12 μM; P3, 15 μM; C2, 120 μM and C8, 100 μM.Figure 2

Dipeptide repeat proteins inhibit homology-directed DNA double strand break repair in C9ORF72 ALS/FTD

BackgroundThe C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated.Methods and resultsUsing DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls.ConclusionsCollectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.

Quantitative Insights into Age-Associated DNA-Repair Inefficiency in Single Cells

SUMMARYAlthough double-strand break (DSB) repair is essential for a cell’s survival, little is known about how DSB repair mechanisms are affected by age. Here we characterize the impact of cellular aging on the efficiency of single-strand annealing (SSA), a DSB repair mechanism. We measure SSA repair efficiency in young and old yeast cells and report a 23.4% decline in repair efficiency. This decline is not due to increased use of non-homologous end joining. Instead, we identify increased G1 phase duration in old cells as a factor responsible for the decreased SSA repair efficiency. Expression of 3xCLN2 leads to higher SSA repair efficiency in old cells compared with expression of 1xCLN2, confirming the involvement of cell-cycle regulation in age-associated repair inefficiency. Examining how SSA repair efficiency is affected by sequence heterology, we find that heteroduplex rejection remains high in old cells. Our work provides insights into the links between single-cell aging and DSB repair efficiency.

Functional Evaluation of CRISPR Activity by the Dual-Fluorescent Surrogate System: C-Check.

Rapid evaluation of the CRISPR gRNA activity is an essential step of employing the technology in editing genes. Through machine learning strategy, the rule sets for in silico designing gRNAs with high activity has greatly improved. However, there are still discrepancies between different prediction rule sets, and between the predicted and actual gRNA activities. Thus, experimentally validating gRNA activity is still the gold standard in defining the best gRNAs for gene editing experiments. One such approach for experimentally selecting gRNAs with high activity is fluorescent surrogate reporter vectors. We had previously developed a dual-fluorescent surrogate system, called C-Check, which based on single-strand annealing repair of the DNA double-strand breaks introduced by CRISPR-Cas9 to generate a functional EGFP. The system offers a tool for rapid functional evaluation of CRISPR gRNA activity, as well as for enrichment of gene edited cells. In this chapter, we will give a step-by-step instruction on the design, generation, and application of the C-Check system for quantifying gRNA activities.

Gene cloning and seamless site-directed mutagenesis using single-strand annealing (SSA)

The full length of interested genes can be usually cloned by assembling exons or RACE products through overlap PCR. However, the procedure requires multiple PCR steps, which are prone to random mutagenesis. Here, we present a novel SSA-based method for gene cloning and seamless site-directed mutagenesis. We firstly cloned the full-length coding sequence of Cashmere goat (Capra hircus) Hoxc13 gene by assembling exons amplified from genomic DNA. Secondly, we created a Hoxc13 loss-function mutant seamlessly and further illustrated that direct repeat length of 25bp is enough to trigger the SSA repair in routine E. coli strains including DH5α, Trans1t1, JM109, and Top10. Moreover, we cloned another full-length mutant of Foxn1 gene from Cashmere goat cDNA using further shortened direct repeats of 19bp. In summary, our study provided an alternative method to overcome the difficulties during overlap PCR in some particular cases for gene cloning.

RNF169 limits 53BP1 deposition at DSBs to stimulate single-strand annealing repair

Unrestrained 53BP1 activity at DNA double-strand breaks (DSBs) hampers DNA end resection and upsets DSB repair pathway choice. RNF169 acts as a molecular rheostat to limit 53BP1 deposition at DSBs, but how this fine balance translates to DSB repair control remains undefined. In striking contrast to 53BP1, ChIP analyses of AsiSI-induced DSBs unveiled that RNF169 exhibits robust accumulation at DNA end-proximal regions and preferentially targets resected, RPA-bound DSBs. Accordingly, we found that RNF169 promotes CtIP-dependent DSB resection and favors homology-mediated DSB repair, and further showed that RNF169 dose-dependently stimulates single-strand annealing repair, in part, by alleviating the 53BP1-imposed barrier to DSB end resection. Our results highlight the interplay of RNF169 with 53BP1 in fine-tuning choice of DSB repair pathways.

Development and application of dual-fluorescence reporter systems for measuring specific nuclease activity based on SSA repair mechanism.

Reporter vector system has become an important method for measuring activity of specific nucleases because of its fast construction, simple modification, easy operation, economic effectiveness as well as its role in enriching positive cells with genomic modification through mediating screen of specific nucleases positive cells. After introducing double strand breaks (DSBs), a reporter system based on non-homologous end joining (NHEJ)-mediated repair can only repair maximally two thirds of reporter genes after optimization, while single strand annealing (SSA)-mediated repair can repair all reporter genes theoretically which has higher sensitivity and facilitates the detection of specific nuclease with low activity and provides an effective way to detect specific nuclease activity in genome modification studies. In this study, we designed and constructed three sets of dual-fluorescence reporter systems based on SSA repair mechanism and applied the mRFP-eGFP system in measuring the effective activity of three pairs of ZFNs, which was 8.9%, 9.3% and 5.0%, respectively. Our study provides an effective way to detect the activity of nucleases.

A novel protein, Rsf1/Pxd1, is critical for the single-strand annealing pathway of double-strand break repair in Schizosaccharomyces pombe.

The process of single-strand annealing (SSA) repairs DNA double-strand breaks that are flanked by direct repeat sequences through the coordinated actions of a series of proteins implicated in recombination, mismatch repair and nucleotide excision repair (NER). Many of the molecular and mechanistic insights gained in SSA repair have principally come from studies in the budding yeast Saccharomyces cerevisiae. However, there is little molecular understanding of the SSA pathway in the fission yeast Schizosaccharomyces pombe. To further our understanding of this important process, we established a new chromosome-based SSA assay in fission yeast. Our genetic analyses showed that, although many homologous components participate in SSA repair in these species indicating that some evolutionary conservation, Saw1 and Slx4 are not principal agents in the SSA repair pathway in fission yeast. This is in marked contrast to the function of Saw1 and Slx4 in budding yeast. Additionally, a novel genus-specific protein, Rsf1/Pxd1, physically interacts with Rad16, Swi10 and Saw1 in vitro and in vivo. We find that Rsf1/Pxd1 is not required for NER and demonstrate that, in fission yeast, Rsf1/Pxd1, but not Saw1, plays a critical role in SSA recombination.