Genome-wide functional screens enable the prediction of high activity CRISPR-Cas9 and -Cas12a guides in Yarrowia lipolytica

Baisya, Dipankar; Ramesh, Adithya; Schwartz, Cory; Lonardi, Stefano; Wheeldon, Ian

doi:10.1038/s41467-022-28540-0

Title: Genome-wide functional screens enable the prediction of high activity CRISPR-Cas9 and -Cas12a guides in Yarrowia lipolytica

Journal Article · Thu Feb 17 00:00:00 EST 2022 · Nature Communications

DOI:https://doi.org/10.1038/s41467-022-28540-0· OSTI ID:1845604

Baisya, Dipankar; Ramesh, Adithya; Schwartz, Cory;

;

Abstract Genome-wide functional genetic screens have been successful in discovering genotype-phenotype relationships and in engineering new phenotypes. While broadly applied in mammalian cell lines and in E. coli , use in non-conventional microorganisms has been limited, in part, due to the inability to accurately design high activity CRISPR guides in such species. Here, we develop an experimental-computational approach to sgRNA design that is specific to an organism of choice, in this case the oleaginous yeast Yarrowia lipolytica . A negative selection screen in the absence of non-homologous end-joining, the dominant DNA repair mechanism, was used to generate single guide RNA (sgRNA) activity profiles for both SpCas9 and LbCas12a. This genome-wide data served as input to a deep learning algorithm, DeepGuide, that is able to accurately predict guide activity. DeepGuide uses unsupervised learning to obtain a compressed representation of the genome, followed by supervised learning to map sgRNA sequence, genomic context, and epigenetic features with guide activity. Experimental validation, both genome-wide and with a subset of selected genes, confirms DeepGuide’s ability to accurately predict high activity sgRNAs. DeepGuide provides an organism specific predictor of CRISPR guide activity that with retraining could be applied to other fungal species, prokaryotes, and other non-conventional organisms.

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Research Organization:: Univ. of California, Riverside, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC); National Science Foundation (NSF)

Grant/Contract Number:: SC0019093; 1706545

OSTI ID:: 1845604

Alternate ID(s):: OSTI ID: 1904369

Journal Information:: Nature Communications, Journal Name: Nature Communications Vol. 13 Journal Issue: 1; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United Kingdom

Language:: English

References (43)

Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System Zetsche, Bernd; Gootenberg, Jonathan S.; Abudayyeh, Omar O. Cell, Vol. 163, Issue 3, p. 759-771 https://doi.org/10.1016/j.cell.2015.09.038	journal	October 2015
Host and Pathway Engineering for Enhanced Lycopene Biosynthesis in Yarrowia lipolytica Schwartz, Cory; Frogue, Keith; Misa, Joshua Frontiers in Microbiology, Vol. 8 https://doi.org/10.3389/fmicb.2017.02233	journal	November 2017
Host-Informed Expression of CRISPR Guide RNA for Genomic Engineering in Komagataella phaffii Dalvie, Neil C.; Leal, Justin; Whittaker, Charles A. ACS Synthetic Biology, Vol. 9, Issue 1 https://doi.org/10.1021/acssynbio.9b00372	journal	December 2019
CRISPR/Cas9 cleavage efficiency regression through boosting algorithms and Markov sequence profiling Peng, Hui; Zheng, Yi; Blumenstein, Michael Bioinformatics, Vol. 34, Issue 18 https://doi.org/10.1093/bioinformatics/bty298	journal	April 2018
Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin Strohkendl, Isabel; Saifuddin, Fatema A.; Gibson, Bryan A. Science Advances, Vol. 7, Issue 11 https://doi.org/10.1126/sciadv.abd6030	journal	March 2021
Guide RNA Engineering Enables Dual Purpose CRISPR-Cpf1 for Simultaneous Gene Editing and Gene Regulation in Yarrowia lipolytica Ramesh, Adithya; Ong, Thomas; Garcia, Jaime A. ACS Synthetic Biology, Vol. 9, Issue 4 https://doi.org/10.1021/acssynbio.9b00498	journal	March 2020
Deep learning improves prediction of CRISPR–Cpf1 guide RNA activity Kim, Hui Kwon; Min, Seonwoo; Song, Myungjae Nature Biotechnology, Vol. 36, Issue 3 https://doi.org/10.1038/nbt.4061	journal	January 2018
Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus Fuller, Kevin K.; Chen, Shan; Loros, Jennifer J. Eukaryotic Cell, Vol. 14, Issue 11 https://doi.org/10.1128/EC.00107-15	journal	November 2015
DeepCRISPR: optimized CRISPR guide RNA design by deep learning Chuai, Guohui; Ma, Hanhui; Yan, Jifang Genome Biology, Vol. 19, Issue 1 https://doi.org/10.1186/s13059-018-1459-4	journal	June 2018
Improved sgRNA design in bacteria via genome-wide activity profiling Guo, Jiahui; Wang, Tianmin; Guan, Changge Nucleic Acids Research, Vol. 46, Issue 14 https://doi.org/10.1093/nar/gky572	journal	June 2018
Genome dependent Cas9/gRNA search time underlies sequence dependent gRNA activity Moreb, E. A.; Lynch, M. D. Nature Communications, Vol. 12, Issue 1 https://doi.org/10.1038/s41467-021-25339-3	journal	August 2021
Combining metabolic engineering and process optimization to improve production and secretion of fatty acids Ledesma-Amaro, Rodrigo; Dulermo, Remi; Niehus, Xochitl Metabolic Engineering, Vol. 38 https://doi.org/10.1016/j.ymben.2016.06.004	journal	November 2016
A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity Jinek, M.; Chylinski, K.; Fonfara, I. Science, Vol. 337, Issue 6096, p. 816-821 https://doi.org/10.1126/science.1225829	journal	June 2012
Optimizing Oleaginous Yeast Cell Factories for Flavonoids and Hydroxylated Flavonoids Biosynthesis Lv, Yongkun; Marsafari, Monireh; Koffas, Mattheos ACS Synthetic Biology, Vol. 8, Issue 11 https://doi.org/10.1021/acssynbio.9b00193	journal	October 2019
Sequence Assembly of Yarrowia lipolytica Strain W29/CLIB89 Shows Transposable Element Diversity Magnan, Christophe; Yu, James; Chang, Ivan PLOS ONE, Vol. 11, Issue 9 https://doi.org/10.1371/journal.pone.0162363	journal	September 2016
Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica Schwartz, Cory; Cheng, Jan-Fang; Evans, Robert Metabolic Engineering, Vol. 55 https://doi.org/10.1016/j.ymben.2019.06.007	journal	September 2019
Engineering xylose utilization in Yarrowia lipolytica by understanding its cryptic xylose pathway Rodriguez, Gabriel M.; Hussain, Murtaza Shabbir; Gambill, Lauren Biotechnology for Biofuels, Vol. 9, Issue 1 https://doi.org/10.1186/s13068-016-0562-6	journal	July 2016
CRISPR–Cas9-enabled genetic disruptions for understanding ethanol and ethyl acetate biosynthesis in Kluyveromyces marxianus Löbs, Ann-Kathrin; Engel, Ronja; Schwartz, Cory Biotechnology for Biofuels, Vol. 10, Issue 1 https://doi.org/10.1186/s13068-017-0854-5	journal	June 2017
sgRNA Scorer 2.0: A Species-Independent Model To Predict CRISPR/Cas9 Activity Chari, Raj; Yeo, Nan Cher; Chavez, Alejandro ACS Synthetic Biology, Vol. 6, Issue 5 https://doi.org/10.1021/acssynbio.6b00343	journal	February 2017
Highly parallel genome variant engineering with CRISPR–Cas9 Sadhu, Meru J.; Bloom, Joshua S.; Day, Laura Nature Genetics, Vol. 50, Issue 4 https://doi.org/10.1038/s41588-018-0087-y	journal	April 2018
The influence of eukaryotic chromatin state on CRISPR–Cas9 editing efficiencies Verkuijl, Sebald AN; Rots, Marianne G. Current Opinion in Biotechnology, Vol. 55 https://doi.org/10.1016/j.copbio.2018.07.005	journal	February 2019
Standardized Markerless Gene Integration for Pathway Engineering in Yarrowia lipolytica Schwartz, Cory; Shabbir-Hussain, Murtaza; Frogue, Keith ACS Synthetic Biology, Vol. 6, Issue 3 https://doi.org/10.1021/acssynbio.6b00285	journal	November 2016
Refined sgRNA efficacy prediction improves large- and small-scale CRISPR–Cas9 applications Labuhn, Maurice; Adams, Felix F.; Ng, Michelle Nucleic Acids Research, Vol. 46, Issue 3 https://doi.org/10.1093/nar/gkx1268	journal	December 2017
The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2020 update Jalili, Vahid; Afgan, Enis; Gu, Qiang Nucleic Acids Research, Vol. 48, Issue 14 https://doi.org/10.1093/nar/gkaa554	journal	June 2020
Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation Gilbert, Luke A.; Horlbeck, Max A.; Adamson, Britt Cell, Vol. 159, Issue 3 https://doi.org/10.1016/j.cell.2014.09.029	journal	October 2014
Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system Liu, Rui; Chen, Ling; Jiang, Yanping Cell Discovery, Vol. 1, Article No. 15007 https://doi.org/10.1038/celldisc.2015.7	journal	May 2015
CRISPR-Mediated Genome Editing and Gene Repression in Scheffersomyces stipitis Cao, Mingfeng; Gao, Meirong; Ploessl, Deon Biotechnology Journal, Vol. 13, Issue 9 https://doi.org/10.1002/biot.201700598	journal	July 2018
Nucleosomes impede Cas9 access to DNA in vivo and in vitro Horlbeck, Max A.; Witkowsky, Lea B.; Guglielmi, Benjamin eLife, Vol. 5 https://doi.org/10.7554/eLife.12677	journal	March 2016
Multiplex Genome Engineering Using CRISPR/Cas Systems Cong, L.; Ran, F. A.; Cox, D. Science, Vol. 339, Issue 6121, p. 819-823 https://doi.org/10.1126/science.1231143	journal	January 2013
Highly Multiplexed CRISPRi Repression of Respiratory Functions Enhances Mitochondrial Localized Ethyl Acetate Biosynthesis in Kluyveromyces marxianus Löbs, Ann-Kathrin; Schwartz, Cory; Thorwall, Sarah ACS Synthetic Biology, Vol. 7, Issue 11 https://doi.org/10.1021/acssynbio.8b00331	journal	October 2018
Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation Doench, John G.; Hartenian, Ella; Graham, Daniel B. Nature Biotechnology, Vol. 32, Issue 12 https://doi.org/10.1038/nbt.3026	journal	September 2014
Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica Xue, Zhixiong; Sharpe, Pamela L.; Hong, Seung-Pyo Nature Biotechnology, Vol. 31, Issue 8 https://doi.org/10.1038/nbt.2622	journal	July 2013
Sequence determinants of improved CRISPR sgRNA design Xu, Han; Xiao, Tengfei; Chen, Chen-Hao Genome Research, Vol. 25, Issue 8 https://doi.org/10.1101/gr.191452.115	journal	June 2015
The Role of Nucleosome Positioning in the Evolution of Gene Regulation Tsankov, Alexander M.; Thompson, Dawn Anne; Socha, Amanda PLoS Biology, Vol. 8, Issue 7 https://doi.org/10.1371/journal.pbio.1000414	journal	July 2010
Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 Doench, John G.; Fusi, Nicolo; Sullender, Meagan Nature Biotechnology, Vol. 34, Issue 2 https://doi.org/10.1038/nbt.3437	journal	January 2016
Nucleosomes inhibit target cleavage by CRISPR-Cas9 in vivo Yarrington, Robert M.; Verma, Surbhi; Schwartz, Shaina Proceedings of the National Academy of Sciences, Vol. 115, Issue 38 https://doi.org/10.1073/pnas.1810062115	journal	September 2018
Synthetic RNA Polymerase III Promoters Facilitate High-Efficiency CRISPR–Cas9-Mediated Genome Editing in Yarrowia lipolytica Schwartz, Cory M.; Hussain, Murtaza Shabbir; Blenner, Mark ACS Synthetic Biology, Vol. 5, Issue 4 https://doi.org/10.1021/acssynbio.5b00162	journal	January 2016
Development of a CRISPR/Cas9-Based Tool for Gene Deletion in Issatchenkia orientalis Tran, Vinh G.; Cao, Mingfeng; Fatma, Zia mSphere, Vol. 4, Issue 3 https://doi.org/10.1128/mSphere.00345-19	journal	June 2019
DeeplyEssential: a deep neural network for predicting essential genes in microbes Hasan, Md Abid; Lonardi, Stefano BMC Bioinformatics, Vol. 21, Issue S14 https://doi.org/10.1186/s12859-020-03688-y	journal	September 2020
Genome and metabolic engineering in non-conventional yeasts: Current advances and applications Löbs, Ann-Kathrin; Schwartz, Cory; Wheeldon, Ian Synthetic and Systems Biotechnology, Vol. 2, Issue 3 https://doi.org/10.1016/j.synbio.2017.08.002	journal	September 2017
CRISPRi repression of nonhomologous end-joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica Schwartz, Cory; Frogue, Keith; Ramesh, Adithya Biotechnology and Bioengineering, Vol. 114, Issue 12 https://doi.org/10.1002/bit.26404	journal	September 2017
Identifying essential genes across eukaryotes by machine learning Beder, Thomas; Aromolaran, Olufemi; Dönitz, Jürgen NAR Genomics and Bioinformatics, Vol. 3, Issue 4 https://doi.org/10.1093/nargab/lqab110	journal	October 2021
Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production Blazeck, John; Hill, Andrew; Liu, Leqian Nature Communications, Vol. 5, Issue 1 https://doi.org/10.1038/ncomms4131	journal	January 2014

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Title: Genome-wide functional screens enable the prediction of high activity CRISPR-Cas9 and -Cas12a guides in Yarrowia lipolytica

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