Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif recognition
While the CRISPR-Cas9 system from S. pyogenes is a powerful genome engineering tool, additional programmed nucleases would enable added flexibility in targeting space and multiplexing. Here, we characterized a CRISPR-Cas9 system from L. gasseri and found that it has modest activity in a cell-free ly...
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| Published in: | PloS one Vol. 13; no. 2; p. e0192181 |
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| Abstract | While the CRISPR-Cas9 system from S. pyogenes is a powerful genome engineering tool, additional programmed nucleases would enable added flexibility in targeting space and multiplexing. Here, we characterized a CRISPR-Cas9 system from L. gasseri and found that it has modest activity in a cell-free lysate assay but no activity in mammalian cells even when altering promoter, position of tag sequences and NLS, and length of crRNA:tracrRNA. In the lysate assay we tested over 400 sequential crRNA target sequences and found that the Lga Cas9 PAM is NNGA/NDRA, different than NTAA predicted from the native bacterial host. In addition, we found multiple instances of consecutive crRNA target sites, indicating flexibility in either PAM sequence or distance from the crRNA target site. This work highlights the need for characterization of new CRISPR systems and highlights the non-triviality of porting them into eukaryotes as gene editing tools. |
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| AbstractList | While the CRISPR-Cas9 system from S. pyogenes is a powerful genome engineering tool, additional programmed nucleases would enable added flexibility in targeting space and multiplexing. Here, we characterized a CRISPR-Cas9 system from L. gasseri and found that it has modest activity in a cell-free lysate assay but no activity in mammalian cells even when altering promoter, position of tag sequences and NLS, and length of crRNA:tracrRNA. In the lysate assay we tested over 400 sequential crRNA target sequences and found that the Lga Cas9 PAM is NNGA/NDRA, different than NTAA predicted from the native bacterial host. In addition, we found multiple instances of consecutive crRNA target sites, indicating flexibility in either PAM sequence or distance from the crRNA target site. This work highlights the need for characterization of new CRISPR systems and highlights the non-triviality of porting them into eukaryotes as gene editing tools. While the CRISPR-Cas9 system from S . pyogenes is a powerful genome engineering tool, additional programmed nucleases would enable added flexibility in targeting space and multiplexing. Here, we characterized a CRISPR-Cas9 system from L . gasseri and found that it has modest activity in a cell-free lysate assay but no activity in mammalian cells even when altering promoter, position of tag sequences and NLS, and length of crRNA:tracrRNA. In the lysate assay we tested over 400 sequential crRNA target sequences and found that the Lga Cas9 PAM is NNGA/NDRA, different than NTAA predicted from the native bacterial host. In addition, we found multiple instances of consecutive crRNA target sites, indicating flexibility in either PAM sequence or distance from the crRNA target site. This work highlights the need for characterization of new CRISPR systems and highlights the non-triviality of porting them into eukaryotes as gene editing tools. |
| Audience | Academic |
| Author | McClelland, Shawn Maksimova, Elena Basila, Megan Smith, Anja van Brabant Barrangou, Rodolphe Briner, Alexandra E Strezoska, Žaklina Anderson, Emily M |
| AuthorAffiliation | 2 Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, United States of America New England Biolabs Inc, UNITED STATES 1 Dharmacon, a Horizon Discovery Group Company, Lafayette, CO, United States of America |
| AuthorAffiliation_xml | – name: 1 Dharmacon, a Horizon Discovery Group Company, Lafayette, CO, United States of America – name: New England Biolabs Inc, UNITED STATES – name: 2 Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, United States of America |
| Author_xml | – sequence: 1 givenname: Emily M surname: Anderson fullname: Anderson, Emily M organization: Dharmacon, a Horizon Discovery Group Company, Lafayette, CO, United States of America – sequence: 2 givenname: Shawn surname: McClelland fullname: McClelland, Shawn organization: Dharmacon, a Horizon Discovery Group Company, Lafayette, CO, United States of America – sequence: 3 givenname: Elena surname: Maksimova fullname: Maksimova, Elena organization: Dharmacon, a Horizon Discovery Group Company, Lafayette, CO, United States of America – sequence: 4 givenname: Žaklina surname: Strezoska fullname: Strezoska, Žaklina organization: Dharmacon, a Horizon Discovery Group Company, Lafayette, CO, United States of America – sequence: 5 givenname: Megan surname: Basila fullname: Basila, Megan organization: Dharmacon, a Horizon Discovery Group Company, Lafayette, CO, United States of America – sequence: 6 givenname: Alexandra E surname: Briner fullname: Briner, Alexandra E organization: Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, United States of America – sequence: 7 givenname: Rodolphe surname: Barrangou fullname: Barrangou, Rodolphe organization: Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, United States of America – sequence: 8 givenname: Anja van Brabant orcidid: 0000-0003-3145-6599 surname: Smith fullname: Smith, Anja van Brabant organization: Dharmacon, a Horizon Discovery Group Company, Lafayette, CO, United States of America |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/29394276$$D View this record in MEDLINE/PubMed |
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| CitedBy_id | crossref_primary_10_15212_bioi_2022_0022 crossref_primary_10_1089_crispr_2019_0068 |
| Cites_doi | 10.1016/j.cell.2015.09.038 10.1099/mic.0.000129 10.1016/j.celrep.2016.09.080 10.1016/j.jbiotec.2015.06.427 10.1016/j.molcel.2016.11.040 10.1016/j.jmb.2015.10.014 10.1101/pdb.top086892 10.1016/j.molcel.2015.10.008 10.1006/meth.2000.1132 10.1016/j.jmb.2016.11.024 10.1016/j.jbiotec.2017.04.017 10.1016/j.cell.2015.11.015 10.7554/eLife.00471 10.1038/nature14299 10.1038/nbt.3659 10.1038/nature13579 10.1038/nbt.3567 10.1038/nature13011 10.1126/science.1231143 10.1016/j.cell.2017.04.005 10.1093/nar/23.14.2677 10.1016/j.molcel.2016.02.031 10.1371/journal.pone.0168968 10.4161/rna.23764 10.1126/science.1258096 10.1126/science.1232033 10.1016/j.molcel.2017.03.016 10.1038/nrmicro.2016.184 |
| ContentType | Journal Article |
| Copyright | COPYRIGHT 2018 Public Library of Science 2018 Anderson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. 2018 Anderson et al 2018 Anderson et al |
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| DOI | 10.1371/journal.pone.0192181 |
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| Notes | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Competing Interests: E.M.A., S.M., E.M., Z.S., M.B., and A.S. were paid employees of GE Healthcare Dharmacon at the time the work was completed. Some of the materials used in the study are products sold by Dharmacon, a Horizon Group company. This does not alter our adherence to all of the PloS ONE publishing ethics. |
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| SubjectTerms | Bacteria Biology and Life Sciences Biotechnology Cell cycle CRISPR Deoxyribonucleic acid DNA Engineering and Technology Eukaryotes Flexibility Genes Genetic modification Genome editing Genomes Genomics Lactobacillus gasseri Mammalian cells Multiplexing Nuclease Nucleases Nutrition Physiological aspects Plasmids Proteins Research and Analysis Methods RNA |
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| Title | Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif recognition |
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