CRISPR-Cas9 technology is an important tool for genome editing because the Cas9 endonuclease can induce targeted DNA double-strand breaks. Targeting of the DNA break is typically controlled by a single-guide RNA (sgRNA) – a chimeric RNA containing a structural segment important for Cas9 binding and a 20mer guide sequence that hybridizes to the genomic DNA target. Previous studies have demonstrated that CRISPR-Cas9 technology can be used for efficient, marker-free genome editing in S. cerevisiae. However, introducing the 20mer guide sequence into yeast sgRNA expression vectors often requires cloning procedures that are complex, time-consuming, and/or expensive. To simplify this process, we have developed a new sgRNA expression cassette with internal restriction enzyme sites that permit rapid, directional cloning of 20mer guide sequences. Here we describe a flexible set of vectors based on this design for cloning and expressing sgRNAs (and Cas9) in yeast using different selectable markers. We anticipate that the Cas9-sgRNA expression vector with the URA3 selectable marker (pML104) will be particularly useful for genome editing in yeast, since the Cas9 machinery can be easily removed by counter-selection using 5-fluoroorotic acid (5-FOA) following successful genome editing. The availability of new vectors that simplify and streamline the technical steps required for guide sequence cloning should help accelerate the use of CRISPR-Cas9 technology in yeast genome editing.

During Cas9 genome editing in eukaryotic cells, the bacterial Cas9 enzyme cleaves DNA targets within chromatin. To understand how chromatin affects Cas9 targeting, we characterized Cas9 activity on nucleosome substrates in vitro. We find that Cas9 endonuclease activity is strongly inhibited when its target site is located within the nucleosome core. In contrast, the nucleosome structure does not affect Cas9 activity at a target site within the adjacent linker DNA. Analysis of target sites that partially overlap with the nucleosome edge indicates that the accessibility of the protospacer-adjacent motif (PAM) is the critical determinant of Cas9 activity on a nucleosome.

DNA damage repair mechanisms have been most thoroughly explored in the eubacterial and eukaryotic branches of life. The methods by which members of the archaeal branch repair DNA are significantly less well understood but have been gaining increasing attention. In particular, the approaches employed by hyperthermophilic archaea have been a general source of interest, since these organisms thrive under conditions that likely lead to constant chromosomal damage. In this work we have characterized the responses of three Sulfolobus solfataricus strains to UV-C irradiation, which often results in double-strand break formation. We examined S. solfataricus strain P2 obtained from two different sources and S. solfataricus strain 98/2, a popular strain for site-directed mutation by homologous recombination. Cellular recovery, as determined by survival curves and the ability to return to growth after irradiation, was found to be strain specific and differed depending on the dose applied. Chromosomal damage was directly visualized using pulsed-field gel electrophoresis and demonstrated repair rate variations among the strains following UV-C irradiation-induced double-strand breaks. Several genes involved in double-strand break repair were found to be significantly upregulated after UV-C irradiation. Transcript abundance levels and temporal expression patterns for doublestrand break repair genes were also distinct for each strain, indicating that these Sulfolobus solfataricus strains have differential responses to UV-C-induced DNA double-strand break damage.Cells have evolved molecular mechanisms to meet the challenge of maintaining genomic integrity by rapidly responding to environmental stresses that can damage proteins and DNA. One of the most common forms of damage is caused by UV light (UV) exposure. High-energy short-wavelength UV-C light is absorbed directly by DNA and induces both cyclobutane pyrimidine dimers between adjacent thymidine or cytosine residues as well as pyrimidine-pyrimidone photoproducts between adjacent pyrimidine residues. Mechanisms for repair of these lesions appear to be present in all organisms and are thought to occur through either light-independent nucleotide excision repair (NER) or light-dependent photoreactivation using photolyases (for reviews, see references 33 and 34). UV-C irradiation also causes the production of reactive oxygen species, which can result in DNA double-strand breaks (DSBs) (6, 44). Our primary understanding for repair of DSBs has come from studies focused primarily on bacteria and eukaryotes. In Escherichia coli, these breaks are repaired through the action of the RecA protein, which assists in recombinational repair of single-strand regions produced through replication fork arrest at UV lesions and in DSB repair by extended synthesis-dependent strand annealing (SDSA) and homologous recombination (9, 19). Eukaryotes employ nonhomologous end joining as well as DSB repair by SDSA and homologous recombination mechanisms to repair these breaks (for recent reviews, see ref...

Cas9 binds and cleaves specific DNA sequences by inducing the formation of an R-loop between the guide RNA and its genomic target. While targeting of active Cas9 to a genomic locus is highly mutagenic because Cas9 creates DNA double strand breaks, targeting of dead Cas9 (dCas9) is presumed not to be mutagenic, as dCas9 lacks DNA endonuclease activity. Here, we show that dCas9 targeting induces mutations in yeast, particularly when targeted to the non-transcribed strand of a gene. dCas9-induced mutations cluster near the guide RNA target region and are comprised of single nucleotide substitutions, small insertions and deletions, and even complex mutations, depending upon the particular guide RNA target. We show that many of these mutations are a consequence of cytosine deamination events occurring on the non-target strand of the dCas9-induced R-loop, while others are associated with homopolymer instability or translesion DNA synthesis. Targeting of dCas9 by a mismatch-containing guide RNA also increases CAN1 mutation frequency, particularly in an ung1Δ mutant strain, suggesting that dCas9 induces mutations through similar mechanisms at off-target sites. These findings indicate that DNA binding by dCas9 is mutagenic in yeast, likely because dCas9 induces the formation of an R-loop at its target site.

Histone posttranslational modifications have been associated with changes in chromatin structure necessary for transcription, replication, and DNA repair. Acetylation is one of the most studied and best characterized histone posttranslational modifications, but it is not known if histone acetylation modulates base excision repair of DNA lesions in chromatin. To address this question, we generated nucleosome core particles (NCPs) containingsite-specificallyacetylatedH3K14orH3K56andmea-sured repair of uracil and single-nucleotide gaps. We find that H3K56Ac and H3K14Ac do not significantly contribute to removal of uracils by uracil DNA glycosylase regardless of the translational or rotational position of the lesions within NCPs. In repair of single-nucleotide gaps, however, the presence of H3K56Ac or H3K14Ac in NCPs decreases the gap-filling activity of DNA polymerase ␤ near the dyad center, with H3K14Ac exhibiting stronger inhibition. To a lesser extent, H3K56Ac induces a similar effect near the DNA ends. Moreover, using restriction enzyme accessibility, we detect no changes in NCP structure or dynamics between H3K14Ac-NCPs and WT-NCPs containing single-nucleotide gaps. Thus, acetylation at H3K56 and H3K14 in nucleosomes may promote alternative gap-filling pathways by inhibiting DNA polymerase ␤ activity.Eukaryotic DNA is organized into arrays of nucleosomes, which constitute the primary level of chromatin compaction. Vital DNA-templated processes, including transcription and DNA repair, are dependent on protein-DNA interactions, which are restricted by the histone proteins in nucleosomes. The nucleosome core particle (NCP) 3 consists of 147 bp of DNA wrapped ϳ1.7 times around a histone octamer composed of a tetramer of histones H3 and H4 ((H3-H4) 2 ) flanked by two heterodimers of histones H2A and H2B (H2A-H2B) (1). Accessibility of nuclear factors to occluded DNA in this stable, yet dynamic protein-DNA complex is regulated by multiple mechanisms, including the activity of chromatin remodeling complexes (2, 3) and the intrinsic, partial unwrapping of DNA ends from the histone octamer (4, 5).The rate of DNA unwrapping, also referred to as DNA breathing, in nucleosomes is dependent on DNA sequence and the posttranslational modification (PTM) status of the histones (6 -8

A critical feature of the intermolecular contacts that bind DNA to the histone octamer is the series of histone arginine residues that insert into the DNA minor groove at each superhelical location where the minor groove faces the histone octamer. One of these "sprocket" arginine residues, histone H4 R45, significantly affects chromatin structure in vivo and is lethal when mutated to alanine or cysteine in Saccharomyces cerevisiae (budding yeast). However, the roles of the remaining sprocket arginine residues (H3 R63, H3 R83, H2A R43, H2B R36, H2A R78, H3 R49) in chromatin structure and other cellular processes have not been well characterized. We have genetically characterized mutations in each of these histone residues when introduced either singly or in combination to yeast cells. We find that pairs of arginine residues that bind DNA adjacent to the DNA exit/entry sites in the nucleosome are lethal in yeast when mutated in combination and cause a defect in histone occupancy. Furthermore, mutations in individual residues compromise repair of UV-induced DNA lesions and affect gene expression and cryptic transcription. This study reveals simple rules for how the location and structural mode of DNA binding influence the biological function of each histone sprocket arginine residue.KEYWORDS nucleotide excision repair; cryptic transcription; nucleosome; histone assembly T HE histone octamer, which is composed of two copies each of histones H2A, H2B, H3, and H4, binds with high affinity to $147 bp of DNA to form the nucleosome core particle. Histone-DNA binding is mediated by .100 histone main-chain and side-chain interactions with the DNA sugarphosphate backbone and a similar number of indirect water-mediated interactions (Davey et al. 2002;Muthurajan et al. 2003). These interactions occur primarily at 14 locations in the nucleosome structure where the DNA minor groove faces the histone octamer [superhelical locations (SHL) 26.5 to 6.5] (Luger et al. 1997). At each of these locations, an arginine side chain extends into the DNA minor groove and makes extensive contacts with the DNA backbone ( Figure 1A).We will refer to the arginine residues that insert into the DNA minor groove as "sprocket" arginines, since they engage the DNA "chain" like the teeth of a bicycle sprocket wheel. Sprocket arginine residues are highly conserved (Muthurajan et al. 2003;Sullivan and Landsman 2003), and the insertion of sprocket arginine side chains into the DNA minor groove comprises a significant fraction of the solvent accessible surface area that is buried upon histone-DNA binding (Davey et al. 2002). Studies have suggested that sprocket arginine-DNA contacts may play an important role in the rotational positioning of nucleosomes (Luger et al. 1997;Harp et al. 2000;Rohs et al. 2009;Wang et al. 2010;West et al. 2010). For example, short poly(A) stretches narrow the DNA minor groove, potentially enhancing electrostatic interactions between the DNA phosphate backbone and the sprocket arginine (Rohs et al. 2009;West et al. 201...

CRISPR‐Cas9 has emerged as a powerful method for editing the genome in a wide variety of species, since it can generate a specific DNA break when targeted by the Cas9‐bound guide RNA. In yeast, Cas9‐targeted DNA breaks are used to promote homologous recombination with a mutagenic template DNA, in order to rapidly generate genome edits (e.g., DNA substitutions, insertions, or deletions) encoded in the template DNA. Since repeated Cas9‐induced DNA breaks select against unedited cells, Cas9 can be used to generate marker‐free genome edits. Here, we describe a simple protocol for constructing Cas9‐expressing plasmids containing a user‐designed guide RNA, as well as protocols for using these plasmids for efficient genome editing in yeast. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Constructing the guide RNA expression vector Basic Protocol 2: Preparing double‐stranded oligonucleotide repair template Alternate Protocol 1: Preparing a single‐stranded oligonucleotide repair template Basic Protocol 3: Induce genome editing by co‐transformation of yeast Basic Protocol 4: Screening for edited cells Basic Protocol 5: Removing sgRNA/CAS9 expression vector Alternate Protocol 2: Removing pML107‐derived sgRNA/CAS9 expression vector

Highlights d Genome sequencing of UV-irradiated yeast reveals noncanonical mutation classes d Non-canonical mutations are likely caused by atypical UV photoproducts d UV induces an atypical thymine-adenine (TA) photoproduct in vitro and in yeast cells d Non-canonical UV mutation classes can explain many driver mutations in melanoma