Homology-Integrated CRISPR–Cas (HI-CRISPR) System for One-Step Multigene Disruption in Saccharomyces cerevisiae
Abstract
One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Bao et al.'s HI-CRISPR system utilized a recyclable plasmid, so that the plasmid can be reused to generate further multiple knockouts in the same strain.
“Furthermore, their new multigene disruption HI-CRISPR system utilized a recyclable plasmid, so that the plasmid can be reused to generate further multiple knockouts in the same strain (Bao et al. 2015).”
From <i>Candida glabrata</i> : new tools and technologies—expanding the toolkit (2015) · cites this paper as Bao et al. 2015
Bao et al. (2015) used the CRISPR-Cas9 system to generate multiple gene disruptions in a single step in S. cerevisiae, representing a powerful tool for creating yeast strains with multiple knockouts in less than a week, termed the HI-CRISPR system.
“Bao et al. (2015) on the other hand have used the CRISPR-Cas9 system to generate multiple gene disruptions in a single step in S. cerevisiae representing a powerful tool for creating yeast strains with multiple knockouts in less than a week, and they have termed this the HI-CRISPR system.”
From <i>Candida glabrata</i> : new tools and technologies—expanding the toolkit (2015) · cites this paper as Bao et al. 2015
Bao et al. (2015) found that triple gene disruptions were also affected by the CRISPR-Cas9 array used.
“In addition, Bao et al. (2015) found that triple gene disruptions were also affected by the CRISPR-Cas9 array (the genome sequence encoding the gRNA strand that directs the degradation/modification of the gene of interest) used; however, this should be addressable in the near future as rules for choosing efficient CRISPR-Cas9 targeting sites emerge with greater understanding of the CRISPR-Cas9”
From <i>Candida glabrata</i> : new tools and technologies—expanding the toolkit (2015) · cites this paper as Bao et al. 2015
Cites
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116 · see citing works
Multiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339 · see citing works
RNA-Guided Human Genome Engineering via Cas9
Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., & Church, G. M. (2013) · Science · doi:10.1126/science.1232033 · Cited by 9,210 · see citing works
Wang, H., Yang, H., Shivalila, C. S., Dawlaty, M. M., Cheng, A. W., Zhang, F., & Jaenisch, R. (2013) · Cell · doi:10.1016/j.cell.2013.04.025 · Cited by 3,495 · see citing works
Efficient genome editing in zebrafish using a CRISPR-Cas system
Hwang, W. Y., Fu, Y., Reyon, D., Maeder, M. L., Tsai, S. Q., Sander, J. D., Peterson, R. T., Yeh, J.-R. J., & Joung, J. K. (2013) · Nature Biotechnology · doi:10.1038/nbt.2501 · Cited by 2,937 · see citing works
Genetic screens in human cells using the CRISPR-Cas9 system
Wang, T., Wei, J. J., Sabatini, D. M., & Lander, E. S. (2014) · Science · doi:10.1126/science.1246981 · Cited by 2,856 · see citing works
High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method
Gietz, R. D., & Schiestl, R. H. (2007) · Nature Protocols · doi:10.1038/nprot.2007.13 · Cited by 2,785 · see citing works
CRISPR RNA Maturation by Trans-Encoded Small RNA and Host Factor RNase III
Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada, Z. A., Eckert, M. R., Vogel, J., & Charpentier, E. (2011) · Nature · doi:10.1038/nature09886 · Cited by 2,563 · see citing works
RNA-guided editing of bacterial genomes using CRISPR-Cas systems
Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013) · Nature Biotechnology · doi:10.1038/nbt.2508 · Cited by 2,429 · see citing works
Crystal structure of Cas9 in complex with guide RNA and target DNA
Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., & Nureki, O. (2014) · Cell · doi:10.1016/j.cell.2014.02.001 · Cited by 2,306 · see citing works
Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems
DiCarlo, J. E., Norville, J. E., Mali, P., Rios, X., Aach, J., & Church, G. M. (2013) · Nucleic Acids Research · doi:10.1093/nar/gkt135 · Cited by 1,566 · see citing works
Foreign gene expression in yeast: a review
Romanos, M. A., Scorer, C. A., & Clare, J. J. (1992) · Yeast · doi:10.1002/yea.320080602 · Cited by 1,046 · see citing works
Metabolic engineering of Escherichia coli for 1-butanol production
Atsumi, S., Cann, A. F., Connor, M. R., Shen, C. R., Smith, K. M., Brynildsen, M. P., Chou, K., Hanai, T., & Liao, J. C. (2007) · Metabolic Engineering · doi:10.1016/j.ymben.2007.08.003 · Cited by 823 · see citing works
Parsons, A. B., Brost, R. L., Ding, H., Li, Z., Zhang, C., Sheikh, B., Brown, G. W., Kane, P. M., Hughes, T. R., & Boone, C. M. (2003) · Nature Biotechnology · doi:10.1038/nbt919 · Cited by 651 · see citing works
CRISPR/Cas System and Its Role in Phage-Bacteria Interactions
Deveau, H., Garneau, J. E., & Moineau, S. (2010) · Annual Review of Microbiology · doi:10.1146/annurev.micro.112408.134123 · Cited by 624 · see citing works
Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism
Komatsu, M., Uchiyama, T., Ōmura, S., Cane, D. E., & Ikeda, H. (2010) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0914833107 · Cited by 502 · see citing works
Metabolic Engineering of Saccharomyces cerevisiae
Østergaard, S., Lisbeth Olsson, A., & Nielsen, J. (2000) · Microbiology and Molecular Biology Reviews · doi:10.1128/mmbr.64.1.34-50.2000 · Cited by 462 · see citing works
Genes required for ionizing radiation resistance in yeast
Bennett, C. B., Lewis, L. K., Karthikeyan, G., Lobachev, K. S., Jin, Y. H., Sterling, J. F., Snipe, J. R., & Resnick, M. A. (2001) · Nature Genetics · doi:10.1038/ng778 · Cited by 328 · see citing works
Total biosynthesis of hydrocortisone from a simple carbon source in yeast
Szczebara, F. M., Chandelier, C., Villeret, C., Heneman-Masurel, A., Bourot, S., Duport, C., Blanchard, S., Groisillier, A., Testet, E., Costaglioli, P., et al. (2003) · Nature Biotechnology · doi:10.1038/nbt775 · Cited by 326 · see citing works
Gene disruption with PCR products in Saccharomyces cerevisiae
Lorenz, M. C., Muir, R. S., Lim, E. Q., McElver, J., Weber, S. C., & Heitman, J. (1995) · Gene · doi:10.1016/0378-1119(95)00144-u · Cited by 286 · see citing works
crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus
Karvelis, T., Gasiūnas, G., Mikšys, A., Barrangou, R., Horvath, P., & Šikšnys, V. (2013) · RNA Biology · doi:10.4161/rna.24203 · Cited by 243 · see citing works
Modelling neurodegeneration in Saccharomyces cerevisiae: why cook with baker's yeast?
Khurana, V., & Lindquist, S. L. (2010) · Nature reviews. Neuroscience · doi:10.1038/nrn2809 · Cited by 235 · see citing works
Raab, A. M., Gebhardt, G., Bolotina, N., Weuster‐Botz, D., & Lang, C. (2010) · Metabolic Engineering · doi:10.1016/j.ymben.2010.08.005 · Cited by 225 · see citing works
THE CAN1 LOCUS OF SACCHAROMYCES CEREVISIAE: FINE-STRUCTURE ANALYSIS AND FORWARD MUTATION RATES
Whelan, W. L., Gocke, E., & Manney, T. R. (1979) · Genetics · doi:10.1093/genetics/91.1.35 · Cited by 156 · see citing works
Transformation of yeast with synthetic oligonucleotides.
Moerschell, R. P., Tsunasawa, S., & Sherman, F. (1988) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.85.2.524 · Cited by 155 · see citing works
Minimization of the Escherichia coli genome using a Tn5-targeted Cre/loxP excision system
Yu, B. J., Sung, B. H., Koob, M. D., Lee, C. H., Lee, J. H., Lee, W. S., Kim, M. S., & Kim, S. C. (2002) · Nature Biotechnology · doi:10.1038/nbt740 · Cited by 145 · see citing works
Exploring redundancy in the yeast genome: an improved strategy for use of the cre–loxP system
Delneri, D., Tomlin, G. C., Wixon, J., Hutter, A., Sefton, M., Louis, E. J., & Oliver, S. G. (2000) · Gene · doi:10.1016/s0378-1119(00)00217-1 · Cited by 127 · see citing works
PCR‐mediated seamless gene deletion and marker recycling in Saccharomyces cerevisiae
Akada, R., Kitagawa, T., Kaneko, S., Toyonaga, D., Ito, S., Kakihara, Y., Hoshida, H., Morimura, S., Kondo, A., & Kida, K. (2006) · Yeast · doi:10.1002/yea.1365 · Cited by 123 · see citing works
New Small Nuclear RNA Gene-Like Transcriptional Units as Sources of Regulatory Transcripts
Pagano, A., Castelnuovo, M., Tortelli, F., et al. (2007) · PLoS Genetics · doi:10.1371/journal.pgen.0030001 · Cited by 102 · see citing works
Donor-recipient mismatch for common gene deletion polymorphisms in graft-versus-host disease
McCarroll, S. A., Bradner, J. E., Turpeinen, H., et al. (2009) · Nature Genetics · doi:10.1038/ng.490 · Cited by 95 · see citing works
Chen, Y., Partow, S., Scalcinati, G., Siewers, V., & Nielsen, J. (2012) · FEMS Yeast Research · doi:10.1111/j.1567-1364.2012.00809.x · Cited by 91 · see citing works
Dorfman, B.-Z. (1969) · Genetics · doi:10.1093/genetics/61.2.377 · Cited by 84 · see citing works
Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection
Suzuki, Y., Onge, R. P. S., Mani, R., King, O. D., Heilbut, A., Labunskyy, V. M., Chen, W., Pham, L., Zhang, L. V., Tong, A. H. Y., et al. (2011) · Nature Methods · doi:10.1038/nmeth.1550 · Cited by 78 · see citing works
Self-citationAnalysis of repair mechanism choice during homologous recombination
Agmon, N., Pur, S., Liefshitz, B., & Kupiec, M. (2009) · Nucleic Acids Research · doi:10.1093/nar/gkp495 · Cited by 73 · see citing works
RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?
Gasiūnas, G., & Šikšnys, V. (2013) · Trends in Microbiology · doi:10.1016/j.tim.2013.09.001 · Cited by 68 · see citing works
Yamamoto, T., Moerschell, R. P., Wakem, P., et al. (1992) · Yeast · doi:10.1002/yea.320081104 · Cited by 66 · see citing works
High level of expression of a protective antigen of schistosomes in Saccharomyces cerevisiae
Loison, G., Vidal, A., Findeli, A., et al. (1989) · Yeast · doi:10.1002/yea.320050609 · Cited by 66 · see citing works
FairyTALE: A High-Throughput TAL Effector Synthesis Platform
Liang, J., Chao, R., Abil, Z., et al. (2013) · ACS Synthetic Biology · doi:10.1021/sb400109p · Cited by 50 · see citing works
Self-citationDoyle, E., Hummel, A. W., Demorest, Z. L., et al. (2013) · PLoS ONE · doi:10.1371/journal.pone.0082120 · Cited by 48 · see citing works
Activities and specificities of homodimeric TALENs in Saccharomyces cerevisiae
Aouida, M., Piatek, M. J., Bangarusamy, D. K., et al. (2013) · Current Genetics · doi:10.1007/s00294-013-0412-z · Cited by 46 · see citing works
Young, L. S., Rivier, D. H., & Sprague, K. U. (1991) · Molecular and Cellular Biology · doi:10.1128/mcb.11.3.1382-1392.1991 · Cited by 26 · see citing works
SunnyTALEN: A second‐generation TALEN system for human genome editing
Sun, N., Bao, Z., Xiong, X., & Zhao, H. (2013) · Biotechnology and Bioengineering · doi:10.1002/bit.25154 · Cited by 24 · see citing works
Self-citationMis-targeting of multiple gene disruption constructs containing hisG
Davidson, J. F., & Schiestl, R. H. (2000) · Current Genetics · doi:10.1007/s002940000154 · Cited by 20 · see citing works
Genetic and Biochemical Properties of Thialysine-resistant Mutants of Saccharomyces cerevisiae
Zwolshen, J. H., & Bhattacharjee, J. K. (1981) · Microbiology · doi:10.1099/00221287-122-2-281 · Cited by 18 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system
Lian, J., HamediRad, M., Hu, S., & Zhao, H. (2017) · Nature Communications · doi:10.1038/s41467-017-01695-x · Cited by 319 · see citing works
Self-citation<i>Candida glabrata</i> : new tools and technologies—expanding the toolkit
Ho, H.-L., & Haynes, K. (2015) · FEMS Yeast Research · doi:10.1093/femsyr/fov066 · Cited by 29 · see citing works
- Authors
- Bao, Z., Xiao, H., Liang, J., Zhang, L., Xiong, X., Sun, N., Si, T., & Zhao, H.
- Affiliations
- University of Illinois Urbana-Champaign (US)
- Corresponding author
- Zhao, Huimin
- Venue
- ACS Synthetic Biology
- Publisher
- American Chemical Society (ACS)
- Published
- 2014-09-19
- Volume
- 4
- Issue
- 5
- Pages
- 585-594
- ISSN
- 2161-5063 (ISSN-L)
- OpenAlex
- W2327297891
- Cited by
- 356
- References
- 44
- Citations by year
- 2026: 10 · 2025: 23 · 2024: 13 · 2023: 24 · 2022: 42 · 2021: 34 · 2020: 39 · 2019: 37 · 2018: 36 · 2017: 42 · 2016: 26 · 2015: 28 · 2014: 2
- FWCI
- 13.40
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- ACS Synthetic Biology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- University of Illinois at Urbana-Champaign
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Keywords
- CRISPR/Cas9, Saccharomyces cerevisiae, homology-directed repair, CAN1, ADE2, ATF2
- MeSH terms
- Base Sequence, Molecular Sequence Data, RNA, Saccharomyces cerevisiae, Amino Acid Transport Systems, Basic, Saccharomyces cerevisiae Proteins, Biosynthetic Pathways, Homologous Recombination, Clustered Regularly Interspaced Short Palindromic Repeats
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
