scholarly journals Optimization of AsCas12a for combinatorial genetic screens in human cells

2019 ◽  
Author(s):  
Kendall R Sanson ◽  
Peter C DeWeirdt ◽  
Annabel K Sangree ◽  
Ruth E Hanna ◽  
Mudra Hegde ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Direct Repeat ◽  
Higher Order ◽  
Human Cells ◽  
Lethal Gene ◽  
Genetic Screens ◽  
Design Rules ◽  
Synthetic Lethal ◽  
Robust System ◽  
Target Design

ABSTRACTCas12a enzymes have attractive properties for scalable delivery of multiplexed perturbations, yet widespread usage has lagged behind Cas9-based strategies. Here we describe the optimization of Cas12a from Acidaminococcus (AsCas12a) for use in pooled genetic screens in human cells. By assaying the activity of thousands of guides, we confirm on-target design rules and extend them to an enhanced activity variant, enAsCas12a. We also develop the first comprehensive set of off-target rules for Cas12a, and demonstrate that we can predict and exclude promiscuous guides. Finally, to enable efficient higher-order multiplexing via lentiviral delivery, we screen thousands of direct repeat variants and identify 38 that outperform the wildtype sequence. We validate this optimized AsCas12a toolkit by targeting 12 synthetic lethal gene pairs with up to 400 guide pairs each, and demonstrate effective triple knockout via flow cytometry. These results establish AsCas12a as a robust system for combinatorial applications of CRISPR technology.

scholarly journals Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network

2014 ◽  
Vol 42 (15) ◽  
pp. 9838-9853 ◽  
Author(s):  
Saeed Kaboli ◽  
Takuya Yamakawa ◽  
Keisuke Sunada ◽  
Tao Takagaki ◽  
Yu Sasano ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Interaction Network ◽  
Essential Genes ◽  
Genetic Interactions ◽  
Lethal Gene ◽  
Synthetic Lethal ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale

Abstract Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

Abstract B09: Combining proteomics and genetic screens to identify KRAS synthetic lethal interactions

2020 ◽  
Author(s):  
Shikha S. Sheth ◽  
Danielle R. Cook ◽  
Samantha D. Strasser ◽  
Timothy D. Martin ◽  
Sneha Menon ◽  
...  
Keyword(s):  
Genetic Screens ◽  
Synthetic Lethal ◽  
Synthetic Lethal Interactions

scholarly journals Fluorescence-Based Phenotypic Selection Allows Forward Genetic Screens in Haploid Human Cells

PLoS ONE ◽  
2012 ◽  
Vol 7 (6) ◽  
pp. e39651 ◽  
Author(s):  
Lidia M. Duncan ◽  
Richard T. Timms ◽  
Eszter Zavodszky ◽  
Florencia Cano ◽  
Gordon Dougan ◽  
...  
Keyword(s):  
Phenotypic Selection ◽  
Human Cells ◽  
Genetic Screens ◽  
Forward Genetic Screens

Synthetic Lethality: From Research to Precision Cancer Nanomedicine

2018 ◽  
Vol 18 (4) ◽  
pp. 337-346 ◽  
Author(s):  
Anuradha Gupta ◽  
Anas Ahmad ◽  
Aqib Iqbal Dar ◽  
Rehan Khan
Keyword(s):  
Cancer Cell ◽  
Mutational Analysis ◽  
Synthetic Lethality ◽  
Therapeutic Index ◽  
Genetic Alterations ◽  
Cancer Drug ◽  
Lethal Gene ◽  
Synthetic Lethal ◽  
Therapeutic Molecules ◽  
Drug Resistant Phenotype

Cancer is an evolutionary disease with multiple genetic alterations, accumulated due to chromosomal instability and/or aneuploidy and it sometimes acquires drug-resistant phenotype also. Whole genome sequencing and mutational analysis helped in understanding the differences among persons for predisposition of a disease and its treatment non-responsiveness. Thus, molecular targeted therapies came into existence. Among them, the concept of synthetic lethality have enthralled great attention as it is a pragmatic approach towards exploiting cancer cell specific mutations to specifically kill cancer cells without affecting normal cells and thus enhancing anti-cancer drug therapeutic index. Thus, this approach helped in discovering new therapeutic molecules for development of precision medicine. Nanotechnology helped in delivering these molecules to the target site in an effective concentration thus reducing off target effects of drugs, dose and dosage frequency drugs. Researchers have tried to deliver siRNA targeting synthetic lethal partner for target cancer cell killing by incorporating it in nanoparticles and it has shown efficacy by preventing tumor progression. This review summarizes the brief introduction of synthetic lethality, and synthetic lethal gene interactions, with a major focus on its therapeutic anticancer potential with the application of nanotechnology for development of personalized medicine.

Identification of Essential Genes and Synthetic Lethal Gene Combinations in Escherichia coli K-12

2015 ◽  
pp. 45-65 ◽  
Author(s):  
Hirotada Mori ◽  
Tomoya Baba ◽  
Katsushi Yokoyama ◽  
Rikiya Takeuchi ◽  
Wataru Nomura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Essential Genes ◽  
Lethal Gene ◽  
Synthetic Lethal ◽  
K 12
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