Deep Learning‐Assisted Automated Single Cell Electroporation Platform for Effective Genetic Manipulation of Hard‐to‐Transfect Cells
Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells....
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| Vydané v: | Small (Weinheim an der Bergstrasse, Germany) Ročník 18; číslo 20; s. e2107795 - n/a |
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Wiley Subscription Services, Inc
01.05.2022
Wiley Blackwell (John Wiley & Sons) |
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| Abstract | Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting for cell isolation that can be harsh to sensitive cell types such as human‐induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work, a fully automated version of the nanofountain probe electroporation (NFP‐E) system, a nanopipette‐based single‐cell electroporation method is presented that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell‐nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP‐E is combined with microconfinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation.
In this article, the authors present a deep learning‐assisted nanofountain probe electroporation system in combination with microconfinement arrays to trap and transfect single cells. Using the combined platform, the authors demonstrate automated intracellular delivery and genetic perturbation in hard‐to‐transfect cells followed by temporal tracking of the perturbed cell colonies. |
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| AbstractList | Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting for cell isolation that can be harsh to sensitive cell types such as human-induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work, a fully automated version of the nanofountain probe electroporation (NFP-E) system, a nanopipette-based single-cell electroporation method is presented that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell-nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP-E is combined with microconfinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation. Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting for cell isolation that can be harsh to sensitive cell types such as human‐induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work, a fully automated version of the nanofountain probe electroporation (NFP‐E) system, a nanopipette‐based single‐cell electroporation method is presented that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell‐nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP‐E is combined with microconfinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation. In this article, the authors present a deep learning‐assisted nanofountain probe electroporation system in combination with microconfinement arrays to trap and transfect single cells. Using the combined platform, the authors demonstrate automated intracellular delivery and genetic perturbation in hard‐to‐transfect cells followed by temporal tracking of the perturbed cell colonies. Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies, is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting (FACS) for cell isolation that can be harsh to sensitive cell types such as human induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work a fully automated version of the nanofountain probe electroporation (NFP-E) system, a nanopipette-based single-cell electroporation method is presented, that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell-nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP-E is combined with micro-confinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation. In this article, the authors present a deep learning assisted Nanofountain Probe Electroporation system in combination with micro-confinement arrays to trap and transfect single cells. Using the combined platform, the authors demonstrate automated intracellular delivery and genetic perturbation in hard-to-transfect cells followed by temporal tracking of the perturbed cell colonies. Abstract Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting for cell isolation that can be harsh to sensitive cell types such as human‐induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work, a fully automated version of the nanofountain probe electroporation (NFP‐E) system, a nanopipette‐based single‐cell electroporation method is presented that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell‐nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP‐E is combined with microconfinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation. Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting for cell isolation that can be harsh to sensitive cell types such as human-induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work, a fully automated version of the nanofountain probe electroporation (NFP-E) system, a nanopipette-based single-cell electroporation method is presented that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell-nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP-E is combined with microconfinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation.Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting for cell isolation that can be harsh to sensitive cell types such as human-induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work, a fully automated version of the nanofountain probe electroporation (NFP-E) system, a nanopipette-based single-cell electroporation method is presented that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell-nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP-E is combined with microconfinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation. |
| Author | Mukherjee, Prithvijit Patino, Cesar A. Pathak, Nibir Lemaitre, Vincent Espinosa, Horacio D. |
| AuthorAffiliation | 3 iNfinitesimal LLC, Skokie, Illinois 60077, United States 1 Department of Mechanical Engineering, Northwestern University, Evanston, Illinois 60208, United States 2 Theoretical and Applied Mechanics Program, Northwestern University, Evanston, Illinois 60208, United States |
| AuthorAffiliation_xml | – name: 1 Department of Mechanical Engineering, Northwestern University, Evanston, Illinois 60208, United States – name: 2 Theoretical and Applied Mechanics Program, Northwestern University, Evanston, Illinois 60208, United States – name: 3 iNfinitesimal LLC, Skokie, Illinois 60077, United States |
| Author_xml | – sequence: 1 givenname: Prithvijit surname: Mukherjee fullname: Mukherjee, Prithvijit organization: iNfinitesimal LLC – sequence: 2 givenname: Cesar A. surname: Patino fullname: Patino, Cesar A. organization: iNfinitesimal LLC – sequence: 3 givenname: Nibir surname: Pathak fullname: Pathak, Nibir organization: Northwestern University – sequence: 4 givenname: Vincent surname: Lemaitre fullname: Lemaitre, Vincent organization: iNfinitesimal LLC – sequence: 5 givenname: Horacio D. orcidid: 0000-0002-1907-3213 surname: Espinosa fullname: Espinosa, Horacio D. email: espinosa@northwestern.edu organization: iNfinitesimal LLC |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/35315229$$D View this record in MEDLINE/PubMed https://www.osti.gov/biblio/1856281$$D View this record in Osti.gov |
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| Keywords | single-cell electroporation deep learning human-induced pluripotent stem cells (hiPSCs) CRISPR/Cas9 intracellular delivery |
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| Notes | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 USDOE equal contribution H.D.E. conceived the project. P.M. and C.A.P. contributed equally. P.M. and C.A.P. wrote the automation software. C.A.P. and P.M. developed the image segmentation routines. C.A.P. and P.M. fabricated the micro-well and micro-pattern arrays. P.M., N.P. and V.L. performed the biological experiments. All authors analyzed and interpreted the data. H.D.E., P.M. and C.A.P wrote the manuscript. Author Contributions |
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| SubjectTerms | Algorithms Automation CRISPR CRISPR-Cas Systems - genetics CRISPR/Cas9 Deep Learning Electroporation Electroporation - methods Gene Editing - methods Genetic modification Humans human‐induced pluripotent stem cells (hiPSCs) Induced Pluripotent Stem Cells - metabolism intracellular delivery Machine learning Nanotechnology Screening single‐cell electroporation Stem cells Workflow |
| Title | Deep Learning‐Assisted Automated Single Cell Electroporation Platform for Effective Genetic Manipulation of Hard‐to‐Transfect Cells |
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