Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9–Cas12a platform

Gonatopoulos-Pournatzis, Thomas; Aregger, Michael; Brown, Kevin R.; Farhangmehr, Shaghayegh; Braunschweig, Ulrich; Ward, Henry N.; Ha, Kevin C.H.; Weiß, Alexander; Billmann, Maximilian; Durbic, Tanja; Myers, Chad L.; Blencowe, Benjamin J.; Moffat, Jason

doi:10.1038/s41587-020-0437-z

Cited by 100 publications

(117 citation statements)

References 84 publications

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“…Synthetic lethality between our hits is corroborated by a dual-gene knockout screen using the CHyMErA hybrid Cas12/Cas9 system [33]. The 678 paralog pairs evaluated in the CHyMErA screens contain 110 pairs targeted in our library, including 12 of the 24 hits we defined.…”

Section: Resultsmentioning

confidence: 96%

Multiplex enCas12a screens detect functional buffering among paralogs otherwise masked in monogenic Cas9 knockout screens

et al. 2020

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Background Pooled library CRISPR/Cas9 knockout screening across hundreds of cell lines has identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens is low compared to the number of constitutively expressed genes in a cell. Results Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observe that half of all constitutively expressed genes are never detected in any CRISPR screen and that these never-essentials are highly enriched for paralogs. We investigated functional buffering among approximately 400 candidate paralog pairs using CRISPR/enCas12a dual-gene knockout screening in three cell lines. We observe 24 synthetic lethal paralog pairs that have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions are present in at least two out of three cell lines and 14 of 24 (58%) are present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes. Conclusions Together, these observations strongly suggest that functionally redundant paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes in monogenic CRISPR-based loss of function screens.

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Section: Resultsmentioning

confidence: 96%

Multiplex enCas12a screens detect functional buffering among paralogs otherwise masked in monogenic Cas9 knockout screens

et al. 2020

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“…Similarly, TIAL1 low expression is associated with TIA1 increased essentiality. Synthetic lethality between these genes is corroborated by a dual-gene knockout screen using a hybrid Cas12/Cas9 system (Gonatopoulos-Pournatzis et al, 2020). Genes CNOT7 and CNOT8 encode alternate subunits of the CCR4-NOT complex, a critical regulator of eukaryotic gene expression 190 (Lau et al, 2009).…”

mentioning

confidence: 99%

Multiplex enCas12a screens show functional buffering by paralogs is systematically absent from genome-wide CRISPR/Cas9 knockout screens

Dede

McLaughlin

Hart³

2020

Preprint

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Major efforts on pooled library CRISPR knockout screening across hundreds of cell lines have identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens are very low compared to the number of constitutively expressed genes in a cell, raising 5 the question of why there are so few essential genes. Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observed that half of all constitutively-expressed genes are never hits in any CRISPR screen, and that these neveressentials are highly enriched for paralogs. We investigated paralog buffering through systematic dual-gene CRISPR knockout screening by testing algorithmically defined ~400 candidate paralog 10 pairs with the enCas12a multiplex knockout system in three cell lines. We observed 24 synthetic lethal paralog pairs which have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions were present in at least two out of three cell lines and 14 of 24 (58%) were present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes. Together these 15 observations strongly suggest that paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes due to genetic buffering in monogenic CRISPR-based mammalian functional genomics approaches. 20 through immense monogenic screening efforts, multiple groups revealed lists of ~2000 highly concordant human essential genes, and comparison of CRISPR technology to orthogonal techniques such as random insertion of gene traps also showed consistent results (Blomen et al., 2015; Hart et al., 2015; Wang et al., 2015).

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“…Positive interactions, on the other hand, can reflect regulatory connections or participation in the same protein complex. Fundamental studies in model organisms have systematically expanded our understanding of genetic regulatory networks and functionally annotated orphan genes 6,7 and recent technological development, in particular the development of CRISPR-based approaches, made now possible to tackle systematic mapping of genetic interaction landscapes in human cells [8][9][10][11][12] .…”

mentioning

confidence: 99%

“…Further studies employing higher-order genetics 10,11,39 or modifications of media composition 40 can be expected to reveal further dependencies among this class of genes. As the functional breadth of SLCs and their potential as a target class becomes increasingly recognized 1,4 , systematic approaches such as the genetic interaction map described here will be necessary to define functions and therapeutically relevant applications of Solute Carriers.…”

mentioning

confidence: 99%

A systematic genetic interaction map of human solute carriers assigns a role to SLC25A51/MCART1 in mitochondrial NAD uptake

Gottardi

Fiume

Goldmann

et al. 2020

Preprint

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Solute Carriers (SLCs) represent the largest family of human transporter proteins, consisting of more than 400 members1,2. Despite the importance of these proteins in determining metabolic states and adaptation to environmental changes, a large proportion of them is still orphan and lacks associated substrates1,3,4. Here we describe a systematic mapping of genetic interactions among SLCs in human cells. Network-based identification of correlated genetic interaction profile neighborhoods resulted in initial functional assignments to dozens of previously uncharacterized SLCs. Focused validation identified SLC25A51/MCART1 as the SLC enabling mitochondrial import of NAD(H). This functional interaction map of the human transportome offers a route for systematic integration of transporter function with metabolism and provides a blueprint for elucidation of the dark genome by biochemical and functional categories.

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Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9–Cas12a platform

Cited by 100 publications

References 84 publications

Multiplex enCas12a screens detect functional buffering among paralogs otherwise masked in monogenic Cas9 knockout screens

Multiplex enCas12a screens detect functional buffering among paralogs otherwise masked in monogenic Cas9 knockout screens

Multiplex enCas12a screens show functional buffering by paralogs is systematically absent from genome-wide CRISPR/Cas9 knockout screens

A systematic genetic interaction map of human solute carriers assigns a role to SLC25A51/MCART1 in mitochondrial NAD uptake

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