Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences

Rybnicky, Grant A.; Fackler, Nicholas A.; Karim, Ashty S.; Köpke, Michael; Jewett, Michael C.

doi:10.1093/nar/gkac142

Title: Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences

Journal Article · Tue Mar 08 00:00:00 UTC 2022 · Nucleic Acids Research

DOI: https://doi.org/10.1093/nar/gkac142 · OSTI ID:1853842

;

Abstract RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a ‘Quick’ method to generate a single PAM prediction or in a ‘Comprehensive’ method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.

View Journal Article

Cite

Export

Save

Research Organization:: Northwestern Univ., Evanston, IL (United States); Northwestern University, Evanston, IL (United States); Univ. of California, Oakland, CA (United States)

Sponsoring Organization:: David and Lucile Packard Foundation; National Science Foundation (NSF); USDOE; USDOE Joint Genome Institute (JGI) Community Science Program; USDOE Office of Science (SC)

Grant/Contract Number:: AC02-05CH11231; SC0018249

OSTI ID:: 1853842

Journal Information:: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 6 Vol. 50; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United Kingdom

Language:: English

References (48)

In Silico Processing of the Complete CRISPR‐Cas Spacer Space for Identification of PAM Sequences Mendoza, Brian J.; Trinh, Cong T. Biotechnology Journal, Vol. 13, Issue 9 https://doi.org/10.1002/biot.201700595	journal	August 2018
Exploiting heterologous and endogenous CRISPR‐Cas systems for genome editing in the probiotic Clostridium butyricum Zhou, Xiuqing; Wang, Xiaolu; Luo, Huiying Biotechnology and Bioengineering, Vol. 118, Issue 7 https://doi.org/10.1002/bit.27753	journal	April 2021
Engineering of vitamin prototrophy in Clostridium ljungdahlii and Clostridium autoethanogenum Annan, Florence J.; Al-Sinawi, Bakir; Humphreys, Christopher M. Applied Microbiology and Biotechnology, Vol. 103, Issue 11 https://doi.org/10.1007/s00253-019-09763-6	journal	April 2019
Basic local alignment search tool Altschul, Stephen F.; Gish, Warren; Miller, Webb Journal of Molecular Biology, Vol. 215, Issue 3, p. 403-410 https://doi.org/10.1016/S0022-2836(05)80360-2	journal	October 1990
Studies on transformation of Escherichia coli with plasmids Hanahan, Douglas Journal of Molecular Biology, Vol. 166, Issue 4 https://doi.org/10.1016/S0022-2836(83)80284-8	journal	June 1983
A novel conjugal donor strain for improved DNA transfer into Clostridium spp. Woods, Craig; Humphreys, Christopher M.; Rodrigues, Raquel Mesquita Anaerobe, Vol. 59 https://doi.org/10.1016/j.anaerobe.2019.06.020	journal	October 2019
Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities van Dongen, Jeanne E.; Berendsen, Johanna T. W.; Steenbergen, Renske D. M. Biosensors and Bioelectronics, Vol. 166 https://doi.org/10.1016/j.bios.2020.112445	journal	October 2020
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
Deciphering, Communicating, and Engineering the CRISPR PAM Leenay, Ryan T.; Beisel, Chase L. Journal of Molecular Biology, Vol. 429, Issue 2 https://doi.org/10.1016/j.jmb.2016.11.024	journal	January 2017
Development of both type I–B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum Walker, Julie E.; Lanahan, Anthony A.; Zheng, Tianyong Metabolic Engineering Communications, Vol. 10 https://doi.org/10.1016/j.mec.2019.e00116	journal	June 2020
CRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity Barrangou, Rodolphe; Marraffini, Luciano A. Molecular Cell, Vol. 54, Issue 2 https://doi.org/10.1016/j.molcel.2014.03.011	journal	April 2014
Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems Leenay, Ryan T.; Maksimchuk, Kenneth R.; Slotkowski, Rebecca A. Molecular Cell, Vol. 62, Issue 1 https://doi.org/10.1016/j.molcel.2016.02.031	journal	April 2016
Metabolic engineering of Clostridium autoethanogenum for selective alcohol production Liew, Fungmin; Henstra, Anne M.; Kӧpke, Michael Metabolic Engineering, Vol. 40 https://doi.org/10.1016/j.ymben.2017.01.007	journal	March 2017
CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum Cho, Jae Sung; Choi, Kyeong Rok; Prabowo, Cindy Pricilia Surya Metabolic Engineering, Vol. 42 https://doi.org/10.1016/j.ymben.2017.06.010	journal	July 2017
Exploiting endogenous CRISPR-Cas system for multiplex genome editing in Clostridium tyrobutyricum and engineer the strain for high-level butanol production Zhang, Jie; Zong, Wenming; Hong, Wei Metabolic Engineering, Vol. 47 https://doi.org/10.1016/j.ymben.2018.03.007	journal	May 2018
A detailed cell-free transcription-translation-based assay to decipher CRISPR protospacer-adjacent motifs Maxwell, Colin S.; Jacobsen, Thomas; Marshall, Ryan Methods, Vol. 143 https://doi.org/10.1016/j.ymeth.2018.02.016	journal	July 2018
Repurposing the Endogenous Type I-E CRISPR/Cas System for Gene Repression in Gluconobacter oxydans WSH-003 Qin, Zhijie; Yang, Yutong; Yu, Shiqin ACS Synthetic Biology, Vol. 10, Issue 1 https://doi.org/10.1021/acssynbio.0c00456	journal	January 2021
High-Level dCas9 Expression Induces Abnormal Cell Morphology in Escherichia coli Cho, Suhyung; Choe, Donghui; Lee, Eunju ACS Synthetic Biology, Vol. 7, Issue 4 https://doi.org/10.1021/acssynbio.7b00462	journal	March 2018
The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA Garneau, Josiane E.; Dupuis, Marie-Ève; Villion, Manuela Nature, Vol. 468, Issue 7320 https://doi.org/10.1038/nature09523	journal	November 2010
In vivo genome editing using Staphylococcus aureus Cas9 Ran, F. Ann; Cong, Le; Yan, Winston X. Nature, Vol. 520, Issue 7546 https://doi.org/10.1038/nature14299	journal	April 2015
Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials Bikard, David; Euler, Chad W.; Jiang, Wenyan Nature Biotechnology, Vol. 32, Issue 11 https://doi.org/10.1038/nbt.3043	journal	October 2014
Applications of CRISPR technologies in research and beyond Barrangou, Rodolphe; Doudna, Jennifer A. Nature Biotechnology, Vol. 34, Issue 9 https://doi.org/10.1038/nbt.3659	journal	September 2016
In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni Kim, Eunji; Koo, Taeyoung; Park, Sung Wook Nature Communications, Vol. 8, Issue 1 https://doi.org/10.1038/ncomms14500	journal	February 2017
Orthogonal Cas9 proteins for RNA-guided gene regulation and editing Esvelt, Kevin M.; Mali, Prashant; Braff, Jonathan L. Nature Methods, Vol. 10, Issue 11 https://doi.org/10.1038/nmeth.2681	journal	September 2013
Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform Rock, Jeremy M.; Hopkins, Forrest F.; Chavez, Alejandro Nature Microbiology, Vol. 2, Issue 4 https://doi.org/10.1038/nmicrobiol.2016.274	journal	February 2017
An updated evolutionary classification of CRISPR–Cas systems Makarova, Kira S.; Wolf, Yuri I.; Alkhnbashi, Omer S. Nature Reviews Microbiology, Vol. 13, Issue 11, p. 722-736 https://doi.org/10.1038/nrmicro3569	journal	September 2015
A thermostable Cas9 with increased lifetime in human plasma Harrington, Lucas B.; Paez-Espino, David; Staahl, Brett T. Nature Communications, Vol. 8, Issue 1 https://doi.org/10.1038/s41467-017-01408-4	journal	November 2017
Carbon-negative production of acetone and isopropanol by gas fermentation at industrial pilot scale Liew, Fungmin Eric; Nogle, Robert; Abdalla, Tanus Nature Biotechnology https://doi.org/10.1038/s41587-021-01195-w	journal	February 2022
In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design Karim, Ashty S.; Dudley, Quentin M.; Juminaga, Alex Nature Chemical Biology, Vol. 16, Issue 8 https://doi.org/10.1038/s41589-020-0559-0	journal	June 2020
Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium Pyne, Michael E.; Bruder, Mark R.; Moo-Young, Murray Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep25666	journal	May 2016
Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus Hidalgo-Cantabrana, Claudio; Goh, Yong Jun; Pan, Meichen Proceedings of the National Academy of Sciences, Vol. 116, Issue 32 https://doi.org/10.1073/pnas.1905421116	journal	July 2019
ggseqlogo: a versatile R package for drawing sequence logos Wagih, Omar Bioinformatics, Vol. 33, Issue 22 https://doi.org/10.1093/bioinformatics/btx469	journal	July 2017
Prophages are associated with extensive CRISPR–Cas auto-immunity Nobrega, Franklin L.; Walinga, Hielke; Dutilh, Bas E. Nucleic Acids Research, Vol. 48, Issue 21 https://doi.org/10.1093/nar/gkaa1071	journal	November 2020
Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C. Nucleic Acids Research, Vol. 44, Issue 5 https://doi.org/10.1093/nar/gkw056	journal	February 2016
PHASTER: a better, faster version of the PHAST phage search tool Arndt, David; Grant, Jason R.; Marcu, Ana Nucleic Acids Research, Vol. 44, Issue W1 https://doi.org/10.1093/nar/gkw387	journal	May 2016
CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins Couvin, David; Bernheim, Aude; Toffano-Nioche, Claire Nucleic Acids Research, Vol. 46, Issue W1 https://doi.org/10.1093/nar/gky425	journal	May 2018
Characterization and repurposing of the endogenous Type I-F CRISPR–Cas system of Zymomonas mobilis for genome engineering Zheng, Yanli; Han, Jiamei; Wang, Baiyang Nucleic Acids Research, Vol. 47, Issue 21 https://doi.org/10.1093/nar/gkz940	journal	October 2019
Transcriptional control of Clostridium autoethanogenum using CRISPRi Fackler, Nicholas; Heffernan, James; Juminaga, Alex Synthetic Biology, Vol. 6, Issue 1 https://doi.org/10.1093/synbio/ysab008	journal	January 2021
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
A Genetic System for Clostridium ljungdahlii: a Chassis for Autotrophic Production of Biocommodities and a Model Homoacetogen Leang, Ching; Ueki, Toshiyuki; Nevin, Kelly P. Applied and Environmental Microbiology, Vol. 79, Issue 4 https://doi.org/10.1128/AEM.02891-12	journal	November 2012
The CRISPR/Cas Adaptive Immune System of Pseudomonas aeruginosa Mediates Resistance to Naturally Occurring and Engineered Phages Cady, K. C.; Bondy-Denomy, J.; Heussler, G. E. Journal of Bacteriology, Vol. 194, Issue 21 https://doi.org/10.1128/JB.01184-12	journal	August 2012
In Vivo Targeting of Clostridioides difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials Selle, Kurt; Fletcher, Joshua R.; Tuson, Hannah mBio, Vol. 11, Issue 2 https://doi.org/10.1128/mBio.00019-20	journal	April 2020
Programmable Removal of Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems Gomaa, Ahmed A.; Klumpe, Heidi E.; Luo, Michelle L. mBio, Vol. 5, Issue 1 https://doi.org/10.1128/mBio.00928-13	journal	January 2014
Stepping on the Gas to a Circular Economy: Accelerating Development of Carbon-Negative Chemical Production from Gas Fermentation Fackler, Nick; Heijstra, Björn D.; Rasor, Blake J. Annual Review of Chemical and Biomolecular Engineering, Vol. 12, Issue 1 https://doi.org/10.1146/annurev-chembioeng-120120-021122	journal	June 2021
Applications of CRISPR Technologies Across the Food Supply Chain Brandt, Katelyn; Barrangou, Rodolphe Annual Review of Food Science and Technology, Vol. 10, Issue 1 https://doi.org/10.1146/annurev-food-032818-121204	journal	March 2019
Genome editing of Clostridium autoethanogenum using CRISPR/Cas9 Nagaraju, Shilpa; Davies, Naomi Kathleen; Walker, David Jeffrey Fraser Biotechnology for Biofuels, Vol. 9, Issue 1 https://doi.org/10.1186/s13068-016-0638-3	journal	October 2016
CRISPR-cas Subtype I-Fb in Acinetobacter baumannii: Evolution and Utilization for Strain Subtyping Karah, Nabil; Samuelsen, Ørjan; Zarrilli, Raffaele PLOS ONE, Vol. 10, Issue 2 https://doi.org/10.1371/journal.pone.0118205	journal	February 2015
CRISPRTarget: Bioinformatic prediction and analysis of crRNA targets Biswas, Ambarish; Gagnon, Joshua N.; Brouns, Stan J. J. RNA Biology, Vol. 10, Issue 5 https://doi.org/10.4161/rna.24046	journal	February 2013

Similar Records

An Archaeal Immune System Can Detect Multiple Protospacer Adjacent Motifs (PAMs) to Target Invader DNA

Journal Article · Thu Jul 05 00:00:00 UTC 2012 · Journal of Biological Chemistry · OSTI ID:1625078

Fischer, Susan; Maier, Lisa-Katharina; Stoll, Britta; +5 more

Characterization and applications of Type I CRISPR-Cas systems

Journal Article · Fri Jan 10 00:00:00 UTC 2020 · Biochemical Society Transactions · OSTI ID:1889422

Hidalgo-Cantabrana, Claudio; Barrangou, Rodolphe

Class 2 CRISPR/Cas compositions and methods of use

Patent · Tue Apr 30 00:00:00 UTC 2024 · OSTI ID:2542101

Doudna, Jennifer A.; Burstein, David; Chen, Janice S.; +3 more

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Biochemistry & Molecular Biology
computational methods
targeted gene modification

Title: Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences

Citation Formats

References (48)

Similar Records

Related Subjects