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| Status |
Public on Jan 28, 2020 |
| Title |
Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform |
| Organisms |
Homo sapiens; Mus musculus |
| Experiment type |
Other
|
| Summary |
Systematic mapping of genetic interactions and interrogation of the functions of sizeable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR-based screening system for combinatorial genetic manipulation that employs co-expression of Cas9 and Cas12a nucleases and machine learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named CHyMErA (Cas Hybrid for Multiplexed Editing and Screening Applications), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive genetic interactions and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemo-genetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for genetic interaction mapping and the functional analysis of sizeable genomic regions, such as alternative exons.
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| Overall design |
Eight CRISPR screens were performed with four different CHyMErA hybird guide (hg)RNA libraries in human and mouse cell lines. All screens were performed with three biologically independent replicates that were derived from the same library infection and were sequenced at multiple time points. (1) A screen were performed using a human optimization hgRNA library in HAP1 cells and that were subjected to 6-thioguanine and thymidine treatment while non-treated cells were used as controls. (2) A screen were performed using a mouse optimization hgRNA library in CGR8 cells. (3) Screens with a human 2nd generation hgRNA library for single- and dual-gene targeting plus paralog pairs were performed in human HAP1 and RPE1 cells. HAP1 cells were also treated with Torin1 and non-treated cells served as control. (4) A screen with a human exon deletion hgRNA library was performed in RPE1 cells.
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| Contributor(s) |
Gonatopoulos-Pournatzis T, Aregger M, Brown KR, Farhangmehr S, Braunschweig U, Ward HN, Ha KC, Weiss A, Billmann M, Durbic T, Myers CL, Blencowe BJ, Moffat J |
| Citation(s) |
32249828 |
| |
| Submission date |
Jan 27, 2020 |
| Last update date |
Apr 28, 2020 |
| Contact name |
Jason Moffat |
| E-mail(s) |
j.moffat@utoronto.ca, jason.moffat@sickkids.ca
|
| Phone |
4379965938
|
| Organization name |
University of Toronto
|
| Department |
Molecular Genetics
|
| Street address |
686 Bay Street, Room 13.9705
|
| City |
Toronto |
| State/province |
ON |
| ZIP/Postal code |
M5G0A4 |
| Country |
Canada |
| |
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| Platforms (3) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
|
| Samples (14)
|
| GSM4284922 |
HAP1 + LbCas12a + Optimization Library |
| GSM4284923 |
HAP1 + AsCas12a + Optimization Library |
| GSM4284924 |
HAP1 + LbCas12a + Optimization Library + Thymidine Block |
| GSM4284925 |
HAP1 + AsCas12a + Optimization Library + Thymidine Block |
| GSM4284926 |
HAP1 + LbCas12a + Optimization Library + 6-thioguanine |
| GSM4284927 |
HAP1 + AsCas12a + Optimization Library + 6-thioguanine |
| GSM4284928 |
N2A + LbCas12a + mouse Optimization Library |
| GSM4284929 |
N2A + AsCas12a + mouse Optimization Library |
| GSM4284930 |
CGR8 + LbCas12a + mouse Optimization Library |
| GSM4284931 |
CGR8 + AsCas12a + mouse Optimization Library |
| GSM4284932 |
HAP1 + LbCas12a + DualTargeting Library |
| GSM4284933 |
HAP1 + LbCas12a + DualTargeting Library + Torin1 |
| GSM4284934 |
RPE1 + LbCas12a + DualTargeting Library |
| GSM4284935 |
RPE1 + LbCas12a + ExonDeletion Library |
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| Relations |
| BioProject |
PRJNA603290 |
| SRA |
SRP245362 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE144281_chymera_sample_key.txt.gz |
7.1 Kb |
(ftp)(http) |
TXT |
| GSE144281_dgCRISPR_merged_human_NovaSeq_counts2.txt.gz |
6.3 Mb |
(ftp)(http) |
TXT |
| GSE144281_dgCRISPR_merged_mouse_NovaSeq_counts.txt.gz |
8.7 Mb |
(ftp)(http) |
TXT |
| GSE144281_exonDeletionLibrary_rawCounts_NovaSeq_18Sept18.txt.gz |
4.0 Mb |
(ftp)(http) |
TXT |
| GSE144281_human_dualTargetingParalog_libV3_annot.txt.gz |
1.7 Mb |
(ftp)(http) |
TXT |
| GSE144281_human_dualTargetingParalog_libV3_guides.fasta.gz |
910.5 Kb |
(ftp)(http) |
FASTA |
| GSE144281_human_exonDeletion_libV13_annot.txt.gz |
2.6 Mb |
(ftp)(http) |
TXT |
| GSE144281_human_exonDeletion_libV13_guides.fasta.gz |
1.2 Mb |
(ftp)(http) |
FASTA |
| GSE144281_human_optimization_libV7_annot.txt.gz |
1.6 Mb |
(ftp)(http) |
TXT |
| GSE144281_human_optimization_libV7_guides.fasta.gz |
1.1 Mb |
(ftp)(http) |
FASTA |
| GSE144281_mouse_optimization_libV3_annot.txt.gz |
1.6 Mb |
(ftp)(http) |
TXT |
| GSE144281_mouse_optimization_libV3_guides.fasta.gz |
1.1 Mb |
(ftp)(http) |
FASTA |
| GSE144281_paralogLibrary_rawCounts_NovaSeq_18Sept18.txt.gz |
4.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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