CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles

Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5′ overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9–mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11’s nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

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CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles

10.1101/193466 ◽

2017 ◽

Cited By ~ 1

Author(s):

Brenda R. Lemos ◽

Adam C. Kaplan ◽

Ji Eun Bae ◽

Alex Ferazzoli ◽

James Kuo ◽

...

Keyword(s):

Dna Polymerase ◽

Mammalian Cells ◽

Budding Yeast ◽

Nuclease Activity ◽

Strand Break ◽

End Joining ◽

Guide Rna ◽

Dna Double Strand Break ◽

Nhej Repair ◽

Dna Strands

AbstractHarnessing CRISPR-Cas9 technology has provided an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We have analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in the budding yeast Saccharomyces cerevisiae and find that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a one base pair (+1) insertion that appears to result from Cas9 creating a 1-bp 5’ overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which were biased toward homonucleotide insertions. Using inverted PAM (iPAM) sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9::gRNA complex influences subsequent NHEJ processing. As with well-studied events induced by the site-specific HO endonuclease, CRISPR-Cas9 mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events, however, are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11’s nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells (van Overbeek et al., 2016) reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

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Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing

10.1101/2021.08.10.455572 ◽

2021 ◽

Author(s):

Martin Peterka ◽

Nina Akrap ◽

Songyuan Li ◽

Sandra Wimberger ◽

Pei-Pei Hsieh ◽

...

Keyword(s):

Mammalian Cells ◽

Strand Break ◽

End Joining ◽

Dsb Repair ◽

Guide Rna ◽

Large Deletions ◽

Dna Double Strand Break ◽

Repair Mechanisms ◽

Non Homologous End Joining ◽

Genomic Insertions

Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two novel strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing lead to increased levels of by-products, it rescued pegRNAs that performed poorly with a nickase-based prime editor. We also present a small molecule approach that yielded increased product purity of PEn editing. Next, we developed a homology-independent PEn editing strategy by engineering a single primed insertion gRNA (springRNA) which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.

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Analysis of DNA Double-Strand Break Repair by Nonhomologous End Joining in Cell-Free Extracts From Mammalian Cells

Molecular Toxicology Protocols ◽

10.1385/1-59259-840-4:351 ◽

2004 ◽

pp. 351-372 ◽

Cited By ~ 1

Author(s):

Petra Pfeiffer ◽

Elke Feldmann ◽

Andrea Odersky ◽

Steffi Kuhfittig-Kulle ◽

Wolfgang Goedecke

Keyword(s):

Mammalian Cells ◽

Strand Break ◽

Double Strand Break ◽

Nonhomologous End Joining ◽

Double Strand Break Repair ◽

End Joining ◽

Dna Double Strand Break ◽

Break Repair

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DNA double-strand break repair within heterochromatic regions

Biochemical Society Transactions ◽

10.1042/bst20110631 ◽

2012 ◽

Vol 40 (1) ◽

pp. 173-178 ◽

Cited By ~ 27

Author(s):

Johanne M. Murray ◽

Tom Stiff ◽

Penny A. Jeggo

Keyword(s):

Chromatin Structure ◽

Mammalian Cells ◽

Strand Break ◽

Higher Order ◽

End Joining ◽

Strand Breaks ◽

Dna Double Strand Break ◽

Dna Dsbs ◽

A Cell ◽

Non Homologous End Joining

DNA DSBs (double-strand breaks) represent a critical lesion for a cell, with misrepair being potentially as harmful as lack of repair. In mammalian cells, DSBs are predominantly repaired by non-homologous end-joining or homologous recombination. The kinetics of repair of DSBs can differ widely, and recent studies have shown that the higher-order chromatin structure can dramatically affect the pathway utilized, the rate of repair and the genetic factors required for repair. Studies of the repair of DSBs arising within heterochromatic DNA regions have provided insight into the constraints that higher-order chromatin structure poses on repair and the processing that is uniquely required for the repair of such DSBs. In the present paper, we provide an overview of our current understanding of the process of heterochromatic DSB repair in mammalian cells and consider the evolutionary conservation of the processes.

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Regulation of the cellular DNA double-strand break response

Biochemistry and Cell Biology ◽

10.1139/o07-135 ◽

2007 ◽

Vol 85 (6) ◽

pp. 663-674 ◽

Cited By ~ 61

Author(s):

Kendra L. Cann ◽

Geoffrey G. Hicks

Keyword(s):

Mammalian Cells ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Breaks ◽

Cell Cycle Checkpoints ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Dna Double Strand Break ◽

Cellular Dna

DNA double-strand breaks occur frequently in cycling cells, and are also induced by exogenous sources, including ionizing radiation. Cells have developed integrated double-strand break response pathways to cope with these lesions, including pathways that initiate DNA repair (either via homologous recombination or nonhomologous end joining), the cell-cycle checkpoints (G1–S, intra-S phase, and G2–M) that provide time for repair, and apoptosis. However, before any of these pathways can be activated, the damage must first be recognized. In this review, we will discuss how the response of mammalian cells to DNA double-strand breaks is regulated, beginning with the activation of ATM, the pinnacle kinase of the double-strand break signalling cascade.

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Protection of Telomeres by the Ku Protein in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.11.10.3265 ◽

2000 ◽

Vol 11 (10) ◽

pp. 3265-3275 ◽

Cited By ~ 97

Author(s):

Peter Baumann ◽

Thomas R. Cech

Keyword(s):

Mammalian Cells ◽

Normal Growth ◽

Strand Break ◽

Telomere Maintenance ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dna Double Strand Break ◽

Ligase Iv ◽

Associated Sequences ◽

Ku Protein

Schizosaccharomyces pombe cells survive loss of telomeres by a unique pathway of chromosome circularization. Factors potentially involved in this survival mechanism include the heterodimeric Ku protein and ligase IV, both of which are involved in the repair of DNA double-strand breaks in mammalian cells. Furthermore, Ku plays a role in telomere maintenance as well as in DNA double-strand break repair in Saccharomyces cerevisiae. We have identified Ku and ligase IV homologues in S. pombe and analyzed their functions during normal growth and in cells undergoing senescence. In the absence of either a Ku subunit (pku70 +) or ligase IV (lig4 +), nonhomologous DNA end-joining was severely reduced. Lack of functional Ku led to shorter but stable telomeres and caused striking rearrangements of telomere-associated sequences, indicating a function for Ku in inhibiting recombinational activities near chromosome ends. In contrast to S. cerevisiae, concurrent deletion ofpku70 + and the gene for the catalytic subunit of telomerase (trt1 +) was not lethal, allowing for the first time the dissection of the roles of Ku during senescence. Our results support a model in which Ku protects chromosome termini from nucleolytic and recombinational activities but is not involved in the formation of chromosome end fusions during senescence. The conclusion that nonhomologous end-joining is not required for chromosome circularization was further supported by analysis of survivors in strains lacking the genes for bothtrt1 + and lig4 +.

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Phosphorylated CtIP bridges DNA to promote annealing of broken ends

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2008645117 ◽

2020 ◽

Vol 117 (35) ◽

pp. 21403-21412

Author(s):

Robin Öz ◽

Sean M. Howard ◽

Rajhans Sharma ◽

Hanna Törnkvist ◽

Ilaria Ceppi ◽

...

Keyword(s):

Nuclease Activity ◽

Strand Break ◽

End Joining ◽

Oligomeric State ◽

Mrn Complex ◽

Nanofluidic Channels ◽

Structural Functions ◽

Dna Double Strand Break ◽

Yeast Homolog ◽

Dna Ends

The early steps of DNA double-strand break (DSB) repair in human cells involve the MRE11-RAD50-NBS1 (MRN) complex and its cofactor, phosphorylated CtIP. The roles of these proteins in nucleolytic DSB resection are well characterized, but their role in bridging the DNA ends for efficient and correct repair is much less explored. Here we study the binding of phosphorylated CtIP, which promotes the endonuclease activity of MRN, to single long (∼50 kb) DNA molecules using nanofluidic channels and compare it to the yeast homolog Sae2. CtIP bridges DNA in a manner that depends on the oligomeric state of the protein, and truncated mutants demonstrate that the bridging depends on CtIP regions distinct from those that stimulate the nuclease activity of MRN. Sae2 is a much smaller protein than CtIP, and its bridging is significantly less efficient. Our results demonstrate that the nuclease cofactor and structural functions of CtIP may depend on the same protein population, which may be crucial for CtIP functions in both homologous recombination and microhomology-mediated end-joining.

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Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.16.8.4189 ◽

1996 ◽

Vol 16 (8) ◽

pp. 4189-4198 ◽

Cited By ~ 180

Author(s):

G T Milne ◽

S Jin ◽

K B Shannon ◽

D T Weaver

Keyword(s):

Saccharomyces Cerevisiae ◽

Mammalian Cells ◽

Strand Break ◽

Yeast Cells ◽

Recombinational Repair ◽

End Joining ◽

Dsb Repair ◽

Dna Double Strand Break ◽

Homologous Recombinational Repair

DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were also sensitive to methylmethane sulfonate (MMS). Ku-dependent repair is distinct from homologous recombination, because deletion of KU80 and HDF1 increased the MMS sensitivity of rad52delta. Interestingly, rad5Odelta, also shown here to be defective in end joining, was epistatic with Ku mutations for MMS repair and end joining. Therefore, Ku and Rad50 participate in an end-joining pathway that is distinct from homologous recombinational repair. Yeast DNA end joining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells.

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Nej1(XLF) inhibits Sae2(CTIP)-Dna2(DNA2) mediated resection at DNA double strand breaks

10.1101/2021.04.10.439283 ◽

2021 ◽

Author(s):

Aditya Mojumdar ◽

Nancy Adam ◽

Jennifer A Cobb

Keyword(s):

Synthetic Lethality ◽

Nuclease Activity ◽

Strand Break ◽

Dna Double Strand Breaks ◽

End Joining ◽

Genomic Deletions ◽

Dna Double Strand Break ◽

Break Site ◽

Dna End Resection ◽

Non Homologous End Joining

The two major pathways of DNA double strand break (DSB) repair, non-homologous end-joining (NHEJ) and homologous recombination (HR), are highly conserved from yeast to mammals. Regulated 5 DNA end resection is important for repair pathway choice and repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends and, more recently, it was shown to be important for DNA end-bridging and inhibiting Dna2-Sgs1 mediated 5 resection. Dna2 localizes to DSBs in the absence of Sgs1 through interactions with Mre11 and Sae2 and DNA damage sensitivity is greater in cells lacking Dna2 nuclease activity compared to sgs1∆ mutants. Dna2-Sae2 mediated 5 resection is down-regulated by Nej1, which itself interacts with Sae2. The resection defect of sae2∆ and the synthetic lethality of sae2∆ sgs1∆ are reversed by deletion of NEJ1 and dependent on Dna2 nuclease activity. Our work demonstrates the importance of Nej1 in inhibiting short-range resection at a DSB by Dna2-Sae2, a critical regulatory mechanism that prevents the formation of genomic deletions at the break site.

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