| Autor: |
Adithya Ramesh, Varun Trivedi, Sangcheon Lee, Aida Tafrishi, Cory Schwartz, Amirsadra Mohseni, Mengwan Li, Stefano Lonardi, Ian Wheeldon |
| Jazyk: |
angličtina |
| Rok vydání: |
2023 |
| Předmět: |
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| Zdroj: |
Communications Biology, Vol 6, Iss 1, Pp 1-11 (2023) |
| Druh dokumentu: |
article |
| ISSN: |
2399-3642 |
| DOI: |
10.1038/s42003-023-04996-8 |
| Popis: |
Abstract High throughput CRISPR screens are revolutionizing the way scientists unravel the genetic underpinnings of engineered and evolved phenotypes. One of the critical challenges in accurately assessing screening outcomes is accounting for the variability in sgRNA cutting efficiency. Poorly active guides targeting genes essential to screening conditions obscure the growth defects that are expected from disrupting them. Here, we develop acCRISPR, an end-to-end pipeline that identifies essential genes in pooled CRISPR screens using sgRNA read counts obtained from next-generation sequencing. acCRISPR uses experimentally determined cutting efficiencies for each guide in the library to provide an activity correction to the screening outcomes via calculation of an optimization metric, thus determining the fitness effect of disrupted genes. CRISPR-Cas9 and -Cas12a screens were carried out in the non-conventional oleaginous yeast Yarrowia lipolytica and acCRISPR was used to determine a high-confidence set of essential genes for growth under glucose, a common carbon source used for the industrial production of oleochemicals. acCRISPR was also used in screens quantifying relative cellular fitness under high salt conditions to identify genes that were related to salt tolerance. Collectively, this work presents an experimental-computational framework for CRISPR-based functional genomics studies that may be expanded to other non-conventional organisms of interest. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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