A novel CRISPR-Cas9 platform with divergent targeting capabilities
Thesis: S.M., Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2018.
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | eng |
| Published: |
Massachusetts Institute of Technology
2018
|
| Subjects: | |
| Online Access: | http://hdl.handle.net/1721.1/119080 |
| _version_ | 1826194795060527104 |
|---|---|
| author | Chatterjee, Pranam |
| author2 | Joseph M. Jacobson. |
| author_facet | Joseph M. Jacobson. Chatterjee, Pranam |
| author_sort | Chatterjee, Pranam |
| collection | MIT |
| description | Thesis: S.M., Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2018. |
| first_indexed | 2024-09-23T10:01:55Z |
| format | Thesis |
| id | mit-1721.1/119080 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T10:01:55Z |
| publishDate | 2018 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/1190802019-04-12T22:57:05Z A novel CRISPR-Cas9 platform with divergent targeting capabilities Novel clustered regularly interspaced short palindromic repeats-associated 9 platform with divergent targeting capabilities Chatterjee, Pranam Joseph M. Jacobson. Program in Media Arts and Sciences (Massachusetts Institute of Technology) Program in Media Arts and Sciences (Massachusetts Institute of Technology) Program in Media Arts and Sciences () Thesis: S.M., Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2018. Cataloged from PDF version of thesis. Includes bibliographical references (pages 47-49). RNA-guided DNA endonucleases of the CRISPR-Cas system are widely used for genome engineering and thus have numerous applications in a wide variety of fields. The range of sequences that CRISPR endonucleases can recognize, however, is constrained by the need for a specific protospacer adjacent motif (PAM) flanking the target site. In this thesis, we demonstrate the natural PAM plasticity of a highly-similar, yet previously uncharacterized, Cas9 from Streptococcus canis (ScCas9) through rational manipulation of distinguishing motif insertions. To this end, we report a divergent affinity to 5'-NNGT-3' PAM sequences, as well as preferences for expanded 5'-NNG- 3' motifs, and demonstrate the editing capabilities of the ortholog in both bacterial and human cells. We subsequently build an automated bioinformatics pipeline, the Search for PAMs by ALignment Of Targets (SPAMALOT), which further explores the microbial PAM diversity of otherwise-overlooked Streptococcus Cas9 orthologs. Our results establish that ScCas9 can be utilized both as an alternative genome editing tool and as a functional platform to discover novel Streptococcus PAM specificities. Finally, we develop original machine learning-based tools to both predict the efficacy of single guide RNA (sgRNA) sequences targeting specific loci, as well as to classify and characterize the recently-discovered anti-CRISPR proteins. by Pranam Chatterjee. S.M. 2018-11-15T16:35:43Z 2018-11-15T16:35:43Z 2018 2018 Thesis http://hdl.handle.net/1721.1/119080 1057896797 eng MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission. http://dspace.mit.edu/handle/1721.1/7582 49 pages application/pdf Massachusetts Institute of Technology |
| spellingShingle | Program in Media Arts and Sciences () Chatterjee, Pranam A novel CRISPR-Cas9 platform with divergent targeting capabilities |
| title | A novel CRISPR-Cas9 platform with divergent targeting capabilities |
| title_full | A novel CRISPR-Cas9 platform with divergent targeting capabilities |
| title_fullStr | A novel CRISPR-Cas9 platform with divergent targeting capabilities |
| title_full_unstemmed | A novel CRISPR-Cas9 platform with divergent targeting capabilities |
| title_short | A novel CRISPR-Cas9 platform with divergent targeting capabilities |
| title_sort | novel crispr cas9 platform with divergent targeting capabilities |
| topic | Program in Media Arts and Sciences () |
| url | http://hdl.handle.net/1721.1/119080 |
| work_keys_str_mv | AT chatterjeepranam anovelcrisprcas9platformwithdivergenttargetingcapabilities AT chatterjeepranam novelclusteredregularlyinterspacedshortpalindromicrepeatsassociated9platformwithdivergenttargetingcapabilities AT chatterjeepranam novelcrisprcas9platformwithdivergenttargetingcapabilities |