publication . Article . Other literature type . 2017

A platform for functional assessment of large variant libraries in mammalian cells.

Matreyek, Kenneth A.; Stephany, Jason J.; Fowler, Douglas M.;
  • Published: 01 Mar 2017
Abstract
Abstract Sequencing-based, massively parallel genetic assays have revolutionized our ability to quantify the relationship between many genotypes and a phenotype of interest. Unfortunately, variant library expression platforms in mammalian cells are far from ideal, hindering the study of human gene variants in their physiologically relevant cellular contexts. Here, we describe a platform for phenotyping variant libraries in transfectable mammalian cell lines in two steps. First, a landing pad cell line with a genomically integrated, Tet-inducible cassette containing a Bxb1 recombination site is created. Second, a single variant from a library of transfected, prom...
Subjects
free text keywords: Methods Online, Genetics, Amino acid, chemistry.chemical_classification, chemistry, Phenotype, Biology, Cell culture, Plasmid
Funded by
NIH| Interdisciplinary Training in Cancer Research Training Grant
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 2T32CA080416-11A1
  • Funding stream: NATIONAL CANCER INSTITUTE
,
NIH| F-CAP: Functionalization of Variants in Clinically Actionable Pharmacogenes
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 5R24GM115277-03
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
,
NIH| Large-Scale Methods for Assessing the Consequences of Mutations in Proteins
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 1R01GM109110-01
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
39 references, page 1 of 3

1.Gasperini M., Starita L., Shendure J.The power of multiplexed functional analysis of ge netic variants. Nat. Protoc.2016; 11:1782–1787.27583640 [OpenAIRE] [PubMed]

2.Shalem O., Sanjana E.N., Hartenian E., Zhang F.Genome-Scale CRISPR-Cas9 Knockout. Science. 2014; 343:84–88.24336571 [OpenAIRE] [PubMed]

3.Wang T., Wei J.J., Sabatini D.M., Lander E.S.Genetic screens in human cells using the CRISPR-Cas9 system. Science. 2014; 343:80–84.24336569 [OpenAIRE] [PubMed]

4.Fowler D.M., Fiel ds S.Deep mutational scanning: a new style of protein science. Nat. Methods. 2014; 11:801–807.25075907 [OpenAIRE] [PubMed]

5.Noderer W.L., Flockhart R.J.Quantitative analysis of mammalian translation initiation sites by FACS‐seq. Mol. Syst. Biol.2014; 10:748.25170020 [OpenAIRE] [PubMed]

6.Duportet X., Wroblewska L., Guye P., Li Y., Eyquem J., Rieders J., Rimchala T., Batt G., Weiss R.A platform for rapid prototyping of synthetic gene networks in mammalian cells. Nuclei c Acids Res.2014; 42:13440–13451.25378321 [OpenAIRE] [PubMed]

7.Schlub T.E., Smyth R.P., Grimm A.J., Mak J., Davenport M.P.Accurately measuring recombination between closely related HIV-1 genomes. PLoS Comput. Biol.2010; 6:e1000766.20442872 [OpenAIRE] [PubMed]

8.Brenan L., Andreev A., Cohen O., Pantel S., Kamburov A., Cacchiarelli D., Persky N.S., Zhu C., Bagul M., Goetz E.M.Phenotypic characterization of a comprehensive set of MAPK1/ERK2 missense mutants. Cell Rep.2016; 17:1171–1183.27760319 [OpenAIRE] [PubMed]

9.Majithia A.R., Tsuda B., Agostini M., Gnanapradeepan K., Rice R., Peloso G., Patel K.A., Zhang X., Broekema M.F., Patterson N.Prospective functional classification of all possible missense variants in PPARG. Nat. Genet.2016; 48:1570–1575.27749844 [OpenAIRE] [PubMed]

10.Findlay G.M., Boyle E.A., Hause R.J., Klein J.C., Shendure J.Saturation editing of genomic regions by multiplex homology-directed repair. Nature. 2014; 513:120–123.25141179 [OpenAIRE] [PubMed]

11.Kumar M., Keller B., Makalou N., Sutton R.E.Systematic determination of the packaging limit of lentiviral vectors. Hum. Gene Ther.2001; 12:1893–905.11589831 [PubMed]

12.He X., Tan C., Wang F., Wang Y., Zhou R., Cui D., You W., Zhao H., Ren J., Feng B.Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair. Nucleic Acids Res.2016; 44:e85.26850641 [OpenAIRE] [PubMed]

13.Lee D., Park J.W., Kim Y., Kim J., Lee Y., Kim J., Kim J.Toward a functional annotation of the human genome using artificial transcription factors. Genome Res.2003; 13:2708–2716.14656973 [OpenAIRE] [PubMed]

14.Xu Z., Thomas L., Davies B., Chalmers R., Smith M., Brown W.Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome. BMC Biotechnol.2013; 13:87.24139482 [OpenAIRE] [PubMed]

15.Yamaguchi S., Kazuki Y., Nakayama Y., Nanba E., Oshimura M., Ohbayashi T.A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector. PLoS One. 2011; 6:e17267.21390305 [OpenAIRE] [PubMed]

39 references, page 1 of 3
Abstract
Abstract Sequencing-based, massively parallel genetic assays have revolutionized our ability to quantify the relationship between many genotypes and a phenotype of interest. Unfortunately, variant library expression platforms in mammalian cells are far from ideal, hindering the study of human gene variants in their physiologically relevant cellular contexts. Here, we describe a platform for phenotyping variant libraries in transfectable mammalian cell lines in two steps. First, a landing pad cell line with a genomically integrated, Tet-inducible cassette containing a Bxb1 recombination site is created. Second, a single variant from a library of transfected, prom...
Subjects
free text keywords: Methods Online, Genetics, Amino acid, chemistry.chemical_classification, chemistry, Phenotype, Biology, Cell culture, Plasmid
Funded by
NIH| Interdisciplinary Training in Cancer Research Training Grant
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 2T32CA080416-11A1
  • Funding stream: NATIONAL CANCER INSTITUTE
,
NIH| F-CAP: Functionalization of Variants in Clinically Actionable Pharmacogenes
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 5R24GM115277-03
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
,
NIH| Large-Scale Methods for Assessing the Consequences of Mutations in Proteins
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 1R01GM109110-01
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
39 references, page 1 of 3

1.Gasperini M., Starita L., Shendure J.The power of multiplexed functional analysis of ge netic variants. Nat. Protoc.2016; 11:1782–1787.27583640 [OpenAIRE] [PubMed]

2.Shalem O., Sanjana E.N., Hartenian E., Zhang F.Genome-Scale CRISPR-Cas9 Knockout. Science. 2014; 343:84–88.24336571 [OpenAIRE] [PubMed]

3.Wang T., Wei J.J., Sabatini D.M., Lander E.S.Genetic screens in human cells using the CRISPR-Cas9 system. Science. 2014; 343:80–84.24336569 [OpenAIRE] [PubMed]

4.Fowler D.M., Fiel ds S.Deep mutational scanning: a new style of protein science. Nat. Methods. 2014; 11:801–807.25075907 [OpenAIRE] [PubMed]

5.Noderer W.L., Flockhart R.J.Quantitative analysis of mammalian translation initiation sites by FACS‐seq. Mol. Syst. Biol.2014; 10:748.25170020 [OpenAIRE] [PubMed]

6.Duportet X., Wroblewska L., Guye P., Li Y., Eyquem J., Rieders J., Rimchala T., Batt G., Weiss R.A platform for rapid prototyping of synthetic gene networks in mammalian cells. Nuclei c Acids Res.2014; 42:13440–13451.25378321 [OpenAIRE] [PubMed]

7.Schlub T.E., Smyth R.P., Grimm A.J., Mak J., Davenport M.P.Accurately measuring recombination between closely related HIV-1 genomes. PLoS Comput. Biol.2010; 6:e1000766.20442872 [OpenAIRE] [PubMed]

8.Brenan L., Andreev A., Cohen O., Pantel S., Kamburov A., Cacchiarelli D., Persky N.S., Zhu C., Bagul M., Goetz E.M.Phenotypic characterization of a comprehensive set of MAPK1/ERK2 missense mutants. Cell Rep.2016; 17:1171–1183.27760319 [OpenAIRE] [PubMed]

9.Majithia A.R., Tsuda B., Agostini M., Gnanapradeepan K., Rice R., Peloso G., Patel K.A., Zhang X., Broekema M.F., Patterson N.Prospective functional classification of all possible missense variants in PPARG. Nat. Genet.2016; 48:1570–1575.27749844 [OpenAIRE] [PubMed]

10.Findlay G.M., Boyle E.A., Hause R.J., Klein J.C., Shendure J.Saturation editing of genomic regions by multiplex homology-directed repair. Nature. 2014; 513:120–123.25141179 [OpenAIRE] [PubMed]

11.Kumar M., Keller B., Makalou N., Sutton R.E.Systematic determination of the packaging limit of lentiviral vectors. Hum. Gene Ther.2001; 12:1893–905.11589831 [PubMed]

12.He X., Tan C., Wang F., Wang Y., Zhou R., Cui D., You W., Zhao H., Ren J., Feng B.Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair. Nucleic Acids Res.2016; 44:e85.26850641 [OpenAIRE] [PubMed]

13.Lee D., Park J.W., Kim Y., Kim J., Lee Y., Kim J., Kim J.Toward a functional annotation of the human genome using artificial transcription factors. Genome Res.2003; 13:2708–2716.14656973 [OpenAIRE] [PubMed]

14.Xu Z., Thomas L., Davies B., Chalmers R., Smith M., Brown W.Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome. BMC Biotechnol.2013; 13:87.24139482 [OpenAIRE] [PubMed]

15.Yamaguchi S., Kazuki Y., Nakayama Y., Nanba E., Oshimura M., Ohbayashi T.A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector. PLoS One. 2011; 6:e17267.21390305 [OpenAIRE] [PubMed]

39 references, page 1 of 3
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publication . Article . Other literature type . 2017

A platform for functional assessment of large variant libraries in mammalian cells.

Matreyek, Kenneth A.; Stephany, Jason J.; Fowler, Douglas M.;