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OPEN A CRISPR‑Cas9 based shufe system for endogenous histone H3 and H4 combinatorial mutagenesis Yu Fu1, Zhenglin Zhu2, Geng Meng1, Rijun Zhang1* & Yueping Zhang1*

Post-translational modifcations of histone greatly impact expression and cell fate decisions in eukaryotes. To study these, it is important to develop a convenient, multiplex, and efcient method to precisely introduce mutations to histones. Because eukaryotic cells usually contain multiple copies of histone , it is a challenge to mutate all histones at the same time by the traditional homologous recombination method. Here, we developed a CRISPR-Cas9 based shufe system in Saccharomyces cerevisiae, to generate point mutations on both endogenous histone H3 and H4 genes in a rapid, seamless and multiplex fashion. Using this method, we generated yeast strains containing histone triple H3–K4R–K36R–K79R mutants and histone combinatorial H3–K56Q–H4– K59A double mutants with high efciencies (70–80%). This CRISPR-Cas9 based mutagenesis system could be an invaluable tool to the epigenetics feld.

In the nucleus of eukaryotic cells, nucleosomes are basic units of chromatin and play essential roles in DNA- templated processes, including packing, transcription, replication, recombination, and repair, together with organization and maintenance of chromatin structure. A nucleosome is composed of about 147 base pairs of DNA wrapped around a histone octamer, which is consist of two copies of core histones H2A, H2B, H3, and ­H41–3. Core histones can be covalently post-translational modifed by methylation, phosphorylation, acetyla- tion, ubiquitylation, and sumoylation. Tese modifcations impact gene expression by either directly altering nucleosome structure or recruiting histone modifers­ 1,4–6. Because there are multiple copies of histone genes in higher eukaryotic organisms, i.e. 100 copies of histone genes in Drosophila melanogaster and 64 copies of his- tone genes in ­ 7,8, it is a challenge to generate histone site-directed mutagenesis to study histone modifcations and related enzymes. Te budding yeast Saccharomyces cerevisiae has been served as a model organism to explore histone functions, because of several advantages (i) its highly-conserved histone sequences and modifcation functions with higher eukaryotes, (ii) its only two copies of histone genes in haploid cell stage, (iii) its ability to survive and grow with only one copy of histone genes, and (iv) its availability of powerful genetic manipulation tools. Several strategies have been carried out for systematic histone mutation libraries in S. cerevisiae: (1) transforming an episomal plasmid containing histone mutants with both endogenous copies deleted, (2) mutating one copy of endogenous histones by homologous recombination (HR) with the other copy deleted, (3) mutating both endogenous copies of histones by HR using two diferent selection ­markers9–11. Tese strategies have been very helpful to study his- tone functions and modifcations, however additional requirements have emerged for new tools. Firstly, histone dosage efects have been discovered for some point mutations, thus previous identifed crucial histone mutants by deleting one or two copies of endogenous histone may have diferent phenotypes with both endogenous cop- ies mutated­ 11,12. Secondly, multiple histone modifcations have the same efect on gene expression or chromatin structure, and methods are needed for generating combinational mutagenesis on histones simultaneously. Last but not least, seamless mutagenesis is preferred for histone mutagenesis to reveal endogenous functions. Te recently developed CRISPR technology has greatly accelerated the speed of genome ­engineering13–16. CRISPR-Cas9 has also been implemented in histone mutagenesis in in a wide range of organisms, including human cell lines, a protozoan parasite Trypanosoma brucei, and Drosophila melanogaster7,17–20. However, there are several limitations for the current CRIPSR-Cas9 system: (i) CRISPR-Cas9 mutagenesis requires an efcient guide RNA (gRNA) targeting sequence and a following “NGG” as Protospacer Adjacent Motif (PAM) which are closed to the mutation sites; however, some targeted mutation sites do not meet such conditions. (ii) Te current

1Laboratory of Feed Biotechnology, State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, College of Veterinary Medicine, China Agricultural University, Haidian District, Beijing 100193, China. 2School of Life Sciences, Chongqing University, No. 55 Daxuecheng South Rd., Shapingba, Chongqing 401331, China. *email: [email protected]; [email protected]

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Chr.II HHT1 HHF1 WT Chr. XIV HHT2 HHF2 Cas9

synH3H4 Chr.II synH3 synH4 master strain Chr. XIV synH3 synH4 Cas9 donor 1 hht1 hhf1

Chr.II synH3 synH4 Cas9 Cas9 Chr. XIV synH3 synH4 Cas9 Cas9 donor 2 hht2 hhf2

hht1, hht2, Chr.II hht1 hhf1 hhf1, hhf2 mutant strain Chr. XIV hht2 hhf2

Figure 1. Graphic representation of CRISPR-Cas9 based shufe system for endogenous histone H3 and H4 mutagenesis. As frst step, synthetic H3 and H4 genes (synH3 and synH4) are shufed into genome by CRISPR- Cas9 to replace endogenous H3 and H4. Ten the synthetic H3 and H4 genes are shufed-out by CRISPR-Cas9 and repaired by donors with endogenous H3 and H4 genes with desired site-directed mutations.

CRISPR-Cas9 systems performed single histone site mutation and are not capable to generate combinatorial histone mutations. In this paper, we developed a CRISPR-Cas9 based shufe system for endogenous histone H3 and H4 combinatorial mutagenesis in Saccharomyces cerevisiae. Using this system, seamless point mutations on histone H3 and H4 in diferent combinations can be generated simultaneously with high efciencies. Results Generating CRISPR‑Cas9 based shufe strains in Saccharomyces cerevisiae. In S. cerevisiae, the core histone genes are organized in H2A–H2B and H3–H4 gene pairs and driven by bidirectional promot- ers. HHT1–HHF1 and HHT2–HHF2 encode histone H3–H4 pairs on II and XIV respectively. Although histone H3 and H4 genes have corresponding identical amino acid sequences, they have 3% (8/309) and 5% (22/409) respectively diferences in DNA coding and dramatically diferent upstream and downstream expression regulating elements. We designed a histone shufe system for endogenous histone H3 and H4 com- binatorial mutagenesis (Fig. 1). Firstly, a shufe master strain was constructed, and endogenous H3 and H4 coding regions were replaced by synthetic H3 and H4 genes (synH3 and synH4) by the CRISPR-Cas9 system. Ten, synH3 and synH4 genes were targeted by CRISPR-Cas9 and repaired by PCR fragments amplifed from endogenous H3 and H4 sequences with desired mutations. To minimize the chance of homology-directed repair at undesired sites, the synH3 and synH4 share same sequences but diferent codons with endogenous H3 and H4 (see sequence alignments in Fig. 2B,C). Te histone shufe master strains with synthetic H3 and H4 (synH3H4) were generated by two steps of CRISPR-Cas9 mediated gene replacement (Fig. 2A). For the frst step, endogenous H3 (HHT1/2) genes are replaced by the synH3 gene. Cas9 plasmids containing 2 gRNAs targeting two loci of H3 genes and synthetic H3 PCR donors with 40–50 bp homologous recombination (HR) arms to the upstream and downstream of both HHT1 and HHT2 genes were electroporated to yeast CEN.PK 113-5D strain. 4 colonies have been randomly selected for verifcation, and 75% of colonies show correct gene replacement at both endogenous loci (Fig. 2A). For the second step, endogenous H4 (HHF1/2) genes are similarly replaced by the synH4 gene with 100% ef- ciency (4/4 colonies) (Fig. 2A). In order to eliminate the possibility that the donor DNA fragments would be integrated into other chromosomal positions with random insertion, we performed whole genome sequencing of synH3H4 strain. And the results showed synH3 and synH4 were integrated at desired loci without any detection of other integration loci (Supplementary data).

Generation of histone H3 single and triple mutant through single transformation. Histone H3 K4, K36, and K79 are the major methylation sites highly associated active gene transcription, and these modifcations are conserved from yeast to human. To determine whether our system can be applied for histone single mutation, we sought out to generate histone H3 K4R mutant frst. Te repairing donors were prepared in two methods: two DNA PCR fragments for repairing and replacing each synH3 gene with K4R mutation sites on the fanking HR arms (total four pieces for hht1 K4R and hht2 K4R) (Fig. 3A), and two single-piece donors containing mutation sites generated by fusion PCR (Fig. 3B). Te donors and plasmids containing Cas9 and 2

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A Chr.II HHT1 HHF1 B gRNA1->HHT1/2 WT HHT1 ATGGCCAGAACAAAGCAAACAGCAAGAAAGTCCACTGGTGGTAAGGCCCCAAGAAAGCAATTAGCTTCTAAGGCTGCCAGAAAATCCGCC Chr. XIV HHT2 HHF2 HHT2 ATGGCCAGAACTAAACAAACAGCTAGAAAATCCACTGGTGGTAAAGCCCCAAGAAAACAATTAGCCTCCAAGGCTGCCAGAAAATCCGCC synH3 ATGGCTAGGACCAAACAGACTGCGAGGAAATCTACCGGCGGAAAGGCTCCCAGGAAACAGTTGGCCTCCAAGGCCGCTAGGAAGTCTGCA Protein MetAlaArgThrLysGlnThrAlaArgLysSerThrGlyGlyLysAlaProArgLysGlnLeuAlaSerLysAlaAlaArgLysSerAla gRNA1->synH3 gRNA2->HHT1/2 HHT1 CCATCTACCGGTGGTGTTAAGAAGCCTCACAGATATAAGCCAGGTACTGTTGCCTTGAGAGAAATTAGAAGATTCCAAAAATCTACTGAA synH3 HHT2 CCATCTACCGGTGGTGTTAAGAAGCCTCACAGATATAAGCCAGGTACTGTTGCCTTGAGAGAAATTAGAAGATTCCAAAAATCTACTGAA synH3 CCCTCCACTGGCGGGGTCAAAAAGCCGCATCGTTACAAACCTGGCACCGTCGCCCTAAGGGAGATTAGGCGTTTTCAGAAGTCCACCGAG Protein ProSerThrGlyGlyValLysLysProHisArgTyrLysProGlyThrValAlaLeuArgGluIleArgArgPheGlnLysSerThrGlu

Chr.II HHT1 HHF1 HHT1 CTGTTGATCAGAAAGTTACCTTTCCAAAGATTGGTCAGAGAAATCGCTCAAGATTTCAAGACCGACTTGAGATTTCAATCTTCTGCCATC Cas9 HHT2 CTGTTGATCAGAAAGTTACCTTTCCAAAGATTGGTCAGAGAAATCGCTCAAGATTTCAAGACCGACTTGAGATTTCAATCTTCTGCTATC Chr. XIV HHT2 HHF2 synH3 CTACTAATAAGGAAACTACCCTTTCAGAGGCTTGTTAGGGAGATTGCGCAGGACTTTAAAACTGATCTACGTTTCCAGTCCTCCGCTATT Protein LeuLeuIleArgLysLeuProPheGlnArgLeuValArgGluIleAlaGlnAspPheLysThrAspLeuArgPheGlnSerSerAlaIle Cas9 gRNA2->synH3 HHT1 GGTGCCTTGCAAGAATCTGTCGAAGCCTACTTAGTCTCTTTATTTGAAGACACCAACTTGGCTGCCATTCACGCCAAGCGTGTCACTATC synH3 HHT2 GGTGCTTTGCAAGAATCCGTCGAAGCATACTTAGTCTCTTTGTTTGAAGACACTAATCTGGCTGCTATTCACGCTAAGCGTGTTACTATC synH3 GGCGCTCTTCAGGAGTCCGTTGAGGCATATTTGGTTTCCTTGTTCGAGGACACTAATCTAGCTGCAATCCATGCTAAAAGGGTTACCATA Protein GlyAlaLeuGlnGluSerValGluAlaTyrLeuValSerLeuPheGluAspThrAsnLeuAlaAlaIleHisAlaLysArgValThrIle

Chr.II synH3 HHF1 HHT1 CAAAAGAAGGATATCAAGTTGGCTAGAAGATTAAGAGGTGAAAGATCATAG HHT2 CAAAAGAAGGATATCAAATTGGCCAGAAGACTAAGAGGTGAAAGATCATGA synH3 synH3 CAGAAGAAAGACATAAAATTAGCCAGGCGTCTGCGTGGCGAGAGGAGCTAG Chr. XIV synH3 HHF2 Protein GlnLysLysAspIleLysLeuAlaArgArgLeuArgGlyGluArgSer*** (efficiency = 3/4) C gRNA1->HHF1/2 HHF1 ATGTCCGGTAGAGGTAAAGGTGGTAAAGGTCTAGGTAAAGGTGGTGCCAAGCGTCACAGAAAGATTCTAAGAGATAACATCCAAGGTATT synH4 HHF2 ATGTCCGGTAGAGGTAAAGGTGGTAAAGGTCTAGGAAAAGGTGGTGCCAAGCGTCACAGAAAGATTCTAAGAGATAACATTCAAGGTATC synH4 ATGTCTGGAAGGGGAAAGGGAGGCAAAGGGTTGGGGAAGGGAGGCGCTAAAAGGCACAGGAAAATATTGAGGGACAATATTCAGGGAATT Protein MetSerGlyArgGlyLysGlyGlyLysGlyLeuGlyLysGlyGlyAlaLysArgHisArgLysIleLeuArgAspAsnIleGlnGlyIle Chr.II synH3 HHF1 gRNA2->HHF1/2 gRNA1->synH4 Cas9 HHF1 ACTAAGCCAGCTATCAGAAGATTAGCTAGAAGAGGTGGTGTCAAGCGTATTTCTGGTTTGATCTACGAAGAAGTCAGAGCTGTCTTGAAA HHF2 ACTAAGCCAGCTATCAGAAGATTAGCTAGAAGAGGTGGTGTCAAGCGTATTTCTGGTTTGATCTACGAAGAAGTCAGAGCCGTCTTGAAA Chr. XIV synH3 HHF2 synH4 ACAAAACCTGCAATTCGTCGTCTAGCACGTAGGGGAGGAGTTAAAAGGATATCCGGATTAATATATGAGGAAGTTAGGGCAGTATTAAAG Cas9 Protein ThrLysProAlaIleArgArgLeuAlaArgArgGlyGlyValLysArgIleSerGlyLeuIleTyrGluGluValArgAlaValLeuLys gRNA2->synH4 synH4 HHF1 TCCTTCTTGGAATCCGTCATCAGAGACTCTGTTACCTACACCGAACACGCCAAGAGAAAGACTGTTACTTCTTTGGATGTTGTTTATGCT HHF2 TCCTTCTTGGAATCCGTCATCAGGGACTCTGTTACTTACACTGAACACGCCAAGAGAAAGACTGTTACTTCTTTGGATGTTGTTTATGCT synH4 TCATTTCTTGAGTCTGTTATAAGGGATTCAGTCACATATACAGAGCATGCAAAAAGGAAAACAGTCACCAGTTTAGACGTCGTCTACGCA Protein SerPheLeuGluSerValIleArgAspSerValThrTyrThrGluHisAlaLysArgLysThrValThrSerLeuAspValValTyrAla

Chr.II synH3 synH4 HHF1 TTGAAGAGACAAGGTAGAACCTTATACGGTTTCGGTGGTTAA synH3H4 HHF2 TTGAAGAGACAAGGTAGAACCTTATATGGTTTCGGTGGTTAA synH4 TTAAAAAGGCAGGGCCGTACATTGTACGGCTTTGGAGGGTAA Chr. XIV synH3 synH4 Protein LeuLysArgGlnGlyArgThrLeuTyrGlyPheGlyGly*** (efficiency = 4/4)

Figure 2. Generation of master shufe strains with synthetic H3 and H4 by CRISPR-Cas9. (A) Generation of a master shufe strain with synthetic H3 and H4 genes (synH3H4) through 2 rounds of CRISPR-Cas9 mediated gene replacements. Te efciencies of 2 rounds are 3/4 (75%) and 4/4 (100%) respectively, which are tested by four randomly picked colonies respectively. (B,C) Te nucleotide sequence alignment of endogenous and synthetic H3 and H4. Te synthetic H3 and H4 (synH3 and synH4) share same protein sequences but diferent DNA codons with native H3 and H4 genes. Te nucleotides with grey background indicate the identical nucleotide sequences. Dark blue arrows represent gRNA sequences targeting endogenous H3 (HHT1/2); brown arrows represent gRNA sequences targeting endogenous H4 (HHF1/2); magenta arrows represent gRNA sequences targeting synH3 genes, and light blue arrows represent gRNA sequences targeting synH4 genes. Te gRNAs targeting synthetic histones are used for generating Histone H3 and H4 mutants in Figs. 3 and 5.

gRNAs targeting synH3 were transformed into the synH3 strain. Te correct gene replacement efciencies of two strategies are at 88% and 71% with no signifcant diference (student T-test, p = 0.1161) (Fig. 3C, lef). Te two strategies for donor preparation were also tested for generating histone H3 K4R–K36R–K79R triple mutant. Te efciency of 79% was achieved by transforming two single-piece donors containing triple mutants, while the replacement efciency of 0% was achieved by using 8-fragment donors with 40–50 bp homologues repair- ing arms (Fig. 3C, right). Te low efciency of the “fragment donors” strategy may be due to the unsuccessful homology direct repair (HDR) of the multiple-fragment donors. To determine whether histone H3 triple mutant sensitive to methyl methanesulfonate (MMS) or hydroxyurea (HU) mediated DNA damage. Serial tenfold dilutions of ­OD600 = 1 were spotted onto YPD plate, YPD plate with 0.05% MMS, or YPD plate with 100 mM HU. And the results show synH3H4 strains are not sensitive to 0.05% MMS or 100 mM HU, while hht1/2 K4R–K36R–K79R mutants are sensitive to 0.05% MMS and 100 mM HU (Fig. 4). Tese results demonstrated that the codon diferences do not afect the shufe master synH3H4 strain sensitive to DNA damage, while the hht1/2 K4R–K36R–K79R mutants are sensitive as previously reported­ 21.

Mutagenesis on both histone H3 and H4 through single transformation. We have demonstrated that the CRISPR-Cas9 based histone shufe system is useful for histone H3 mutagenesis with high efcien- cies. Next, we sought out to determine whether this system can be applied for mutagenesis on both histone H3 and H4 with diferent mutation combinations (Fig. 5A,B). Histone H3 K56Q mutant and histone H4 K59A mutant have been previously shown growth defects on medium containing 200 mM HU compared to wild-type ­strains22. We implemented our system for generating H3 K56Q and H4 K59A on both copies of histone genes or by combination of mutants and wild-type genes (Fig. 5A,B). We frst transformed single-piece donors with Cas9 plasmid containing 2 gRNAs (One gRNA targeting synH3 and the other targeting synH4). To our surprise, there was no successful gene replacement colony (Fig. 5C, lef). Tis result suggested that either DNA cleavaged by Cas9 or HDR may not be efcient. To improve the chance of Cas9 cleavage, the Cas9 plasmid containing 4 gRNAs (with each histone synthetic gene targeted by 2 gRNAs, and gRNA sequences showed in Fig. 2B,C) was constructed and transformed with the same donors. With the help of additional gRNAs, the correct mutant efciencies were improved to 71% for hht1/2 K56Q, hhf1/2 K59A mutant and 75% for hht1 K56Q/HHT2, HHF1/ hhf2 K59A single-copy mutant. Te HU sensitivity assay showed the mutations of hht1/2 K56Q, hhf1/2 K59A

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Figure 3. Generation of histone H3 single and triple mutants through a single transformation. (A) Graphic representation of CRISPR-Cas9 based mutagenesis on histone H3. Te transformation was performed on synH3 strain with 2 targeting gRNAs and donors harboring mutation sites and HR regions. Te single piece donors were generated through fusion PCR. (B) Te mutagenesis efciencies for generating histone H3 single and triple mutant through transformation with diferent formats of donors. Te repairing donors can be prepared by either multiple donor fragments with fanking HR arms for mutation or two single pieces of donors containing mutation sites. Te data of bar charts represent mean averages of over-all mutagenesis efciencies of 3 biological replicates with each randomly picked 8 colonies. Te error bars indicate the standard deviations of 3 biological replicates. (C) Te representation of sequencing results of both Histone H3 K4R–K36R–K79R at endogenous loci. Te sequence alignments were performed by SnapGene sofware.

were sensitive to 100 mM HU, while the single-copy mutants hht1 K56Q/ HHT2, HHF1/ hhf2 K59A were less sensitive to 100 mM HU than double-copy mutants (Fig. 5D). Tese results demonstrated CRISPR-Cas9 based histone shufe system is versatile for mutagenesis of histone H3 and H4 mutations with diferent combinations. Discussions In this study, we developed a convenient, seamless, and efcient CRISPR-Cas9 based shufe system to precisely introduce multiple mutations to histones H3 and H4 in Saccharomyces cerevisiae. We demonstrated this method by generating yeast histone triple H3–K4R–K36R–K79R mutants and histone H3–K56Q–H4–K59A double mutants with high efciencies (70–80%). Tis system is also capable to mutate two copies of histones with any combinatorial mutations. Although CRISPR-Cas9 has been applied to generate protein point mutations, it still needs to consider the limitation of gRNA closed to the desired mutation sites. Our method provides an alternative two-step method:

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Figure 4. Confrmation of MMS and HU sensitivity of synH3H4 strain and histone H3 K4R–K36R, K79R mutant. WT stain and two biological replicates of each mutant were grown in YPD overnight at 30 °C. Serial tenfold dilutions of OD­ 600 = 1 yeast cultures were spotted onto YPD for 2 days, YPD with 0.05% MMS for 3 days, or YPD with 100 mM HU and incubated for 3 days at 30 °C.

A B hht1 * *hhf1 hht1 * HHF1

Chr.II synH3 synH4 Chr.II synH3 synH4 synH3H4 Cas9 Cas9 synH3H4 Cas9 Cas9 Chr.XIV synH3 synH4 Chr.XIV synH3 synH4 Cas9 Cas9 Cas9 Cas9 hht2 * *hhf2 HHT2 *hhf2

hht1, hht2, Chr.II hht1 * *hhf1 hht1, HHT2, Chr.II hht1 * HHF1 hhf1, hhf2 HHF1,hhf2 Chr.XIV hht2 * *hhf2 Chr.XIV HHT2 *hhf2

C 2 gRNAs targeting 4 gRNAs targeting D synH3 and synH4 synH3 and synH4 100%

80% WT ficiency ef 60% hht1/2 K56Q, 40% hhf1/2 K59A

20% hht1 K56Q / HHT2, 0% HHF1 / hhf2 K59A Mutagenesis hht1/2 1234K56Q, hht1 K56Q, hht1/2 K56Q, hht1 K56Q, hhf1/2 K59A HHT2, hhf1/2 K59A HHT2, YPD 100mM HU HHF1, HHF1, hhf2 K59A hhf2 K59A

Figure 5. Mutagenesis on both histone H3 and H4 through single transformation. (A,B) Graphic representation of CRISPR-Cas9 based gene replacement for mutagenesis on both histone H3 and H4 with diferent combinations. By transforming diferent donors, the endogenous two copies of histone H3 and H4 can be repaired for identical mutants (A) or with diferent mutation sites (B). (C) Te mutagenesis efciencies for generating histone H3 and H4 mutants through single transformation with diferent gRNAs. 2 gRNAs: 1 gRNA targeting synH3 and 1 gRNA targeting synH4 and 4 gRNAs: 2 gRNAs for each synthetic histone. Te data of bar charts represent mean averages of over-all mutagenesis efciencies of 3 biological replicates with each randomly picked 8 colonies. Te error bars indicate the standard deviations of 3 biological replicates. (D) Confrmation of HU sensitivity of histone H3 and H4 mutant. Two biological replicates were grown in YPD overnight at 30 °C. Serial tenfold dilutions were spotted onto YPD or YPD with 100 mM HU and incubated at 30 °C for 3 days.

frstly, replacing the original gene with a synthetic copy (codons changed) by CRISPR/Cas9, then targeting the synthetic copy using CRISPR/Cas9 and repair back to the endogenous gene with desired mutations. We also found that increasing gRNA numbers to target a multiple-copy gene can improve gene replacement efciencies. One possible explanation is that a single double strain break (DSB) site would be easily repaired by the other uncleavaged gene copies, while multiple DSB sites on single gene would trigger homology directed repair by donor DNA fragments when other gene copies also cleavaged. Tis fnding can provide strategies for other multiple-copy gene editing. For the future studies, it is useful to generate synthetic master strain with histone H2A, H2B, H3, and H4. Tus, any combinatorial mutations on histone genes can be generated through a single transformation.

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Material and methods Strains and broth media. Escherichia coli Top10 was used for vector cloning. Te yeast strain used in this work was Saccharomyces cerevisiae CEN.PK 113-5D (MAT a MAL2-8c SUC2 ura3-52). E. coli top10 cells were grown on LB-Agar or LB broth with 50 mg/L Amp antibiotics for plasmid construction. Yeast strains were grown in YPD media with 2% glucose before the transformation. Transformants were plated on synthetic complete (SC) media minus uracil plates to select yeast cells. SC-5-FOA agar plates were employed to drop-out Cas9 plasmids.

Plasmid construction. Te SynH3 and synH4 coding sequences were designed according to the codon usage of S. cerevisiae, synthesized by Sangon Biotech (Shanghai) Co., Ltd., and cloned into pUC57 vector. Te pCas vector with multiple gRNA was constructed as the previous report­ 23.

Donor DNA preparation. Donors used in this work were obtained by PCR reaction and purifed by gel extraction. we cloned HHT1–HHF1, and HHT2–HHF2 pair genes with their upstream and downstream sequences on pUC18 vectors. Te repair donors were generated by PCR or fusion PCR with indicated primers amplifcation from the plasmid and purifed by gel extraction.

Yeast transformation and mutant identifcation. Yeast transformation was carried out using the elec- troporation as the previous ­report23. Genome DNA of strains survived on the auxotrophic plate was extracted for PCR reaction. Te genotype of H3 and H4 of mutants were identifed with PCR amplifcation and followed DNA sequencing. And genome DNA of Saccharomyces cerevisiae CEN.PK 113-5D was used as a control. H3 and H4 sequences of mutants were amplifed by primers on ~ 100 bp upper and lower sites for sequencing analysis. Te mutation rate was calculated as the ratio of mutants to total colonies tested with 3 biological replicates.

DNA damage sensitivity assays. WT stain and two biological replicates of each mutant were grown in YPD overnight at 30 °C. Yeast culture with diluted at OD­ 600 = 1 as the starting point. Serial tenfold dilutions of yeast culture were spotted onto YPD for 2 days, YPD with 0.05% MMS for 3 days, or YPD with 100 mM HU and incubated for 3 days at 30 °C.

Received: 20 June 2020; Accepted: 7 January 2021

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23. Zhang, Y. et al. A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae. Nat. Commun. 10(1), 1053 (2019). Acknowledgements Tis work was supported by Outstanding Talent Introduction Program from College of Veterinary Medicine, China Agricultural University, Beijing 100193, China, National Key Research and Development Program of China (2018YFD0500600), National Natural Science Foundation of China (31572442 and 31200941), and the Fundamental Research Funds for the Central Universities (106112016CDJXY290002). Author contributions Y.F., G.M., R.Z. and Y.Z. designed the research; Y.F. carried out the experiment; Y.F., Z.Z, G.M. and Y.Z. analyzed data; Y.Z. wrote the paper. G.M., R.Z. and Y.Z. supervised the research.

Competing interests Te authors declare no competing interests. Additional information Supplementary Information Te online version contains supplementary material available at https​://doi. org/10.1038/s4159​8-021-82774​-4. Correspondence and requests for materials should be addressed to R.Z. or Y.Z. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afliations. Open Access Tis 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 licence, and indicate if changes were made. Te images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/.

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