Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs
MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges,...
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| Language: | English |
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Frontiers Media S.A.
2024-04-01
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| Series: | Frontiers in Molecular Biosciences |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2023.1295507/full |
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| author | Smitha Ijee Smitha Ijee Karthik Chambayil Karthik Chambayil Anurag Dutta Chaudhury Anurag Dutta Chaudhury Abhirup Bagchi Kirti Modak Kirti Modak Saswati Das Saswati Das Esther Sathya Bama Benjamin Esther Sathya Bama Benjamin Sonam Rani Sonam Rani Daniel Zechariah Paul Daniel Zechariah Paul Aneesha Nath Debanjan Roy Debanjan Roy Dhavapriya Palani Sweety Priyanka Rakshini Ravichandran Betty K. Kumary Yazhini Sivamani Vijayanand S. Dinesh Babu Yukio Nakamura Vasanth Thamodaran Vasanth Thamodaran Poonkuzhali Balasubramanian Shaji R. Velayudhan Shaji R. Velayudhan |
| author_facet | Smitha Ijee Smitha Ijee Karthik Chambayil Karthik Chambayil Anurag Dutta Chaudhury Anurag Dutta Chaudhury Abhirup Bagchi Kirti Modak Kirti Modak Saswati Das Saswati Das Esther Sathya Bama Benjamin Esther Sathya Bama Benjamin Sonam Rani Sonam Rani Daniel Zechariah Paul Daniel Zechariah Paul Aneesha Nath Debanjan Roy Debanjan Roy Dhavapriya Palani Sweety Priyanka Rakshini Ravichandran Betty K. Kumary Yazhini Sivamani Vijayanand S. Dinesh Babu Yukio Nakamura Vasanth Thamodaran Vasanth Thamodaran Poonkuzhali Balasubramanian Shaji R. Velayudhan Shaji R. Velayudhan |
| author_sort | Smitha Ijee |
| collection | DOAJ |
| description | MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges, present limitations in terms of specificity, transient effects, and off-target effects. Similarly, CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study, we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration, this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition, we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus, enabling homogeneous, temporal, and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods, the dgRNA-based approach offers higher specificity, allowing for the deletion of individual miRNAs with similar seed sequences, without affecting other miRNAs. Due to the increased design space, the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines, demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation, and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes, resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters, allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes. |
| first_indexed | 2024-04-24T15:09:30Z |
| format | Article |
| id | doaj.art-5312775f75e844e48f5507f1e9c2a7d8 |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-04-24T15:09:30Z |
| publishDate | 2024-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-5312775f75e844e48f5507f1e9c2a7d82024-04-02T11:52:59ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2024-04-011010.3389/fmolb.2023.12955071295507Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAsSmitha Ijee0Smitha Ijee1Karthik Chambayil2Karthik Chambayil3Anurag Dutta Chaudhury4Anurag Dutta Chaudhury5Abhirup Bagchi6Kirti Modak7Kirti Modak8Saswati Das9Saswati Das10Esther Sathya Bama Benjamin11Esther Sathya Bama Benjamin12Sonam Rani13Sonam Rani14Daniel Zechariah Paul15Daniel Zechariah Paul16Aneesha Nath17Debanjan Roy18Debanjan Roy19Dhavapriya Palani20Sweety Priyanka21Rakshini Ravichandran22Betty K. Kumary23Yazhini Sivamani24Vijayanand S.25Dinesh Babu26Yukio Nakamura27Vasanth Thamodaran28Vasanth Thamodaran29Poonkuzhali Balasubramanian30Shaji R. Velayudhan31Shaji R. Velayudhan32Centre for Stem Cell Research (A Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, IndiaDepartment of Biotechnology, Thiruvalluvar University, Vellore, IndiaCentre for Stem Cell Research (A Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, IndiaSree Chitra Tirunal Institute of Science and Medical Technology, Thiruvananthapuram, IndiaDepartment of Haematology, Christian Medical College Campus, Vellore, IndiaRegional Centre for Biotechnology, New Delhi, IndiaCentre for Stem Cell Research (A Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, IndiaDepartment of Haematology, Christian Medical College Campus, Vellore, IndiaRegional Centre for Biotechnology, New Delhi, IndiaDepartment of Biotechnology, Thiruvalluvar University, Vellore, IndiaDepartment of Haematology, Christian Medical College Campus, Vellore, IndiaSree Chitra Tirunal Institute of Science and Medical Technology, Thiruvananthapuram, IndiaDepartment of Haematology, Christian Medical College Campus, Vellore, IndiaCentre for Stem Cell Research (A Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, IndiaDepartment of Biotechnology, Thiruvalluvar University, Vellore, IndiaDepartment of Haematology, Christian Medical College Campus, Vellore, IndiaManipal Academy of Higher Education, Manipal, IndiaCentre for Stem Cell Research (A Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, IndiaDepartment of Haematology, Christian Medical College Campus, Vellore, IndiaManipal Academy of Higher Education, Manipal, IndiaCentre for Stem Cell Research (A Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, IndiaDepartment of Haematology, Christian Medical College Campus, Vellore, IndiaDepartment of Haematology, Christian Medical College Campus, Vellore, IndiaDepartment of Haematology, Christian Medical College Campus, Vellore, IndiaDepartment of Haematology, Christian Medical College Campus, Vellore, IndiaDepartment of Biotechnology, Thiruvalluvar University, Vellore, IndiaCentre for Stem Cell Research (A Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, IndiaCell Engineering Division, RIKEN BioResource Research Center, Tsukuba, JapanCentre for Stem Cell Research (A Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, IndiaTata Institute of Genetics and Society, Bengaluru, IndiaDepartment of Haematology, Christian Medical College Campus, Vellore, IndiaCentre for Stem Cell Research (A Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, IndiaDepartment of Haematology, Christian Medical College Campus, Vellore, IndiaMicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges, present limitations in terms of specificity, transient effects, and off-target effects. Similarly, CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study, we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration, this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition, we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus, enabling homogeneous, temporal, and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods, the dgRNA-based approach offers higher specificity, allowing for the deletion of individual miRNAs with similar seed sequences, without affecting other miRNAs. Due to the increased design space, the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines, demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation, and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes, resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters, allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes.https://www.frontiersin.org/articles/10.3389/fmolb.2023.1295507/fullmiRNACRISPR-Cas9dual-gRNAiPSCserythroid |
| spellingShingle | Smitha Ijee Smitha Ijee Karthik Chambayil Karthik Chambayil Anurag Dutta Chaudhury Anurag Dutta Chaudhury Abhirup Bagchi Kirti Modak Kirti Modak Saswati Das Saswati Das Esther Sathya Bama Benjamin Esther Sathya Bama Benjamin Sonam Rani Sonam Rani Daniel Zechariah Paul Daniel Zechariah Paul Aneesha Nath Debanjan Roy Debanjan Roy Dhavapriya Palani Sweety Priyanka Rakshini Ravichandran Betty K. Kumary Yazhini Sivamani Vijayanand S. Dinesh Babu Yukio Nakamura Vasanth Thamodaran Vasanth Thamodaran Poonkuzhali Balasubramanian Shaji R. Velayudhan Shaji R. Velayudhan Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs Frontiers in Molecular Biosciences miRNA CRISPR-Cas9 dual-gRNA iPSCs erythroid |
| title | Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs |
| title_full | Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs |
| title_fullStr | Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs |
| title_full_unstemmed | Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs |
| title_short | Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs |
| title_sort | efficient deletion of micrornas using crispr cas9 with dual guide rnas |
| topic | miRNA CRISPR-Cas9 dual-gRNA iPSCs erythroid |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2023.1295507/full |
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