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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 31, 2023 |
| Title |
Functional annotation of genetic variants using locally haploid human pluripotent stem cells |
| Organism |
Homo sapiens |
| Experiment type |
Other
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| Summary |
Mutations in the BRCA2 gene are associated with sporadic and familial cancer, cause genomic instability, and sensitize cancer cells to PARP inhibition. We have developed a human primary cell system to annotate variants in BRCA2 and to test the variants’ sensitivities to PARP inhibition. We engineered locally haploid human pluripotent stem cells (loHAPs) that contain a localized deletion encompassing one copy of BRCA2. Next, we characterized essential regions of the BRCA2 gene to identify permissive and loss-of-function mutations in hPSCs and differentiated fibroblasts, using CRISPR-Cas9 editing of the functional allele. Additionally, we used gene editing to directly test the function of individual amino acids, including clinical variants of uncertain significance in BRCA2, and identify alleles that are sensitive PARP inhibitors, a standard of care in BRCA2-deficient cancers. Collectively, our analysis demonstrates that loHAPs can facilitate detailed structure-function analysis of genes and the rapid functional evaluation of clinically-observed mutations.
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| Overall design |
The CRISPR-mediated mutation tiling experients were designed to evaluate the scale and resolution that loHAPs can be employed to annotate BRCA2 function. Each sgRNA was individually delivered into 1 million hPSCs as RNP. After nucleofection, cells were combined into different pools by the position of sgRNA they received (6 sgRNAs in exon 2, 22 sgRNA in exon 27), then each cell pool was seeded onto three 15cm MEF dishes as biological triplicates. At least 0.3 million cells were replated for expansion or collected as genomic DNA every 7 days for 3 weeks. Drug sensitivity screening was started on day 21 with at least 0.3 million cells were used in each replicate, and cells were collected on day 28. At least 2µg phenol-chloroform extracted genomic DNA from each sample was used as a template in PCR amplification of the mutagenized region, then amplicons were purified, barcoded, and NGS-sequenced at depth >1 million reads per sample on PE250 NovaSeq or PE150 NextSeq2000. NGS reads were first processed using CRISPResso2 to trim off adapter sequences, aligned to a provided reference sequence, and filtered by quality.
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| Contributor(s) |
Li H, Hockemeyer D |
| Citation(s) |
37488236 |
| Submission date |
May 30, 2023 |
| Last update date |
Mar 07, 2024 |
| Contact name |
Dirk Hockemeyer |
| E-mail(s) |
hockemeyer@berkeley.edu
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| Phone |
510-664-9851
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| Organization name |
University of California, Berkeley
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| Department |
Department of Molecular & Cell Biology
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| Street address |
400 Li Ka Shing Center
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| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720-3370 |
| Country |
USA |
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| Platforms (2) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
| GPL30173 |
NextSeq 2000 (Homo sapiens) |
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| Samples (63)
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| Relations |
| BioProject |
PRJNA977591 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE233683_RAW.tar |
12.0 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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