Evaluation of CRISPR-Cas9 approaches to investigate microRNA targeting in human chondrocytes
<p>Micro RNAs (miRNAs) are small, non-coding, negative regulators of genes that play a major role in a wide variety of biological and pathological processes. The chondrocyte-selective miR-140 is required for normal cartilage development, as demonstrated by the developmental defects in miR-140...
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
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2019
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| _version_ | 1797112742224592896 |
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| author | Chaudhry, NM |
| author2 | Vincent, T |
| author_facet | Vincent, T Chaudhry, NM |
| author_sort | Chaudhry, NM |
| collection | OXFORD |
| description | <p>Micro RNAs (miRNAs) are small, non-coding, negative regulators of genes that play a major role in a wide variety of biological and pathological processes. The chondrocyte-selective miR-140 is required for normal cartilage development, as demonstrated by the developmental defects in miR-140 knockout (KO) mice. However, only a few studies have been conducted to explore the mechanism of action of miR-140 at the cellular level.</p>
<p>In the present study, we describe a CRISPR-Cas9 system to identify functional miR-140 targets in isolated primary human osteoarthritic articular chondrocytes. CRISPR-Cas9 editing offers a highly target-specific and stable method for exploring functional miRNA targets, without perturbing other miRNA levels. Developing a systematic approach of double transfection using CRISPR-Cas9, we produced rapid, efficient, and reproduceable genetically engineered populations of primary human articular chondrocytes, without prior need for clonal selection.</p>
<p>Stable knockdown of miR-140 (> 90 %), with no detectable off-target activity, identified several miR-140 regulated targets, of which Septin 2 (SEPT2), bone morphogenetic protein 2 (BMP2), and fibroblast growth factor 2 (FGF2) had previously been reported. The major aggrecanase ADAMTS-5 was not a miR-140 target in our study, even though this had been reported previously. A number of novel targets were identified including Agrin (AGRN), a gene encoding for a heparan sulfate proteoglycan with chondrogenic potential, two regulators of the retinoic acid pathway, retinoic acid receptor gamma
(RARG) and cytochrome P450 26B1 (CYP26B1), and two molecules critical for primary cilium function, intraflagellar transport 88 (IFT88) and Tau Tubulin Kinase 2 (TTBK2).</p>
<p>In order to attempt to differentiate between direct and indirect effects of the miRNA, I adopted a novel CRISPR-Ca9 based technology, in which the miRNA response element (MRE) of the target gene, rather than the miRNA itself, was targeted. I report the challenges associated with this approach in primary human chondrocytes.</p>
<p>Taken together, I describe a highly efficient and reproduceable CRISPR-Cas9-mediated protocol, in which I identified novel miR-140 targets of disease relevance using primary OA chondrocytes.</p> |
| first_indexed | 2024-03-07T08:29:43Z |
| format | Thesis |
| id | oxford-uuid:c60741e5-aca2-410c-ab3d-9e97bead4107 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T08:29:43Z |
| publishDate | 2019 |
| record_format | dspace |
| spelling | oxford-uuid:c60741e5-aca2-410c-ab3d-9e97bead41072024-02-29T08:37:51ZEvaluation of CRISPR-Cas9 approaches to investigate microRNA targeting in human chondrocytesThesishttp://purl.org/coar/resource_type/c_db06uuid:c60741e5-aca2-410c-ab3d-9e97bead4107EnglishORA Deposit2019Chaudhry, NMVincent, T<p>Micro RNAs (miRNAs) are small, non-coding, negative regulators of genes that play a major role in a wide variety of biological and pathological processes. The chondrocyte-selective miR-140 is required for normal cartilage development, as demonstrated by the developmental defects in miR-140 knockout (KO) mice. However, only a few studies have been conducted to explore the mechanism of action of miR-140 at the cellular level.</p> <p>In the present study, we describe a CRISPR-Cas9 system to identify functional miR-140 targets in isolated primary human osteoarthritic articular chondrocytes. CRISPR-Cas9 editing offers a highly target-specific and stable method for exploring functional miRNA targets, without perturbing other miRNA levels. Developing a systematic approach of double transfection using CRISPR-Cas9, we produced rapid, efficient, and reproduceable genetically engineered populations of primary human articular chondrocytes, without prior need for clonal selection.</p> <p>Stable knockdown of miR-140 (> 90 %), with no detectable off-target activity, identified several miR-140 regulated targets, of which Septin 2 (SEPT2), bone morphogenetic protein 2 (BMP2), and fibroblast growth factor 2 (FGF2) had previously been reported. The major aggrecanase ADAMTS-5 was not a miR-140 target in our study, even though this had been reported previously. A number of novel targets were identified including Agrin (AGRN), a gene encoding for a heparan sulfate proteoglycan with chondrogenic potential, two regulators of the retinoic acid pathway, retinoic acid receptor gamma (RARG) and cytochrome P450 26B1 (CYP26B1), and two molecules critical for primary cilium function, intraflagellar transport 88 (IFT88) and Tau Tubulin Kinase 2 (TTBK2).</p> <p>In order to attempt to differentiate between direct and indirect effects of the miRNA, I adopted a novel CRISPR-Ca9 based technology, in which the miRNA response element (MRE) of the target gene, rather than the miRNA itself, was targeted. I report the challenges associated with this approach in primary human chondrocytes.</p> <p>Taken together, I describe a highly efficient and reproduceable CRISPR-Cas9-mediated protocol, in which I identified novel miR-140 targets of disease relevance using primary OA chondrocytes.</p> |
| spellingShingle | Chaudhry, NM Evaluation of CRISPR-Cas9 approaches to investigate microRNA targeting in human chondrocytes |
| title | Evaluation of CRISPR-Cas9 approaches to investigate microRNA targeting in human chondrocytes |
| title_full | Evaluation of CRISPR-Cas9 approaches to investigate microRNA targeting in human chondrocytes |
| title_fullStr | Evaluation of CRISPR-Cas9 approaches to investigate microRNA targeting in human chondrocytes |
| title_full_unstemmed | Evaluation of CRISPR-Cas9 approaches to investigate microRNA targeting in human chondrocytes |
| title_short | Evaluation of CRISPR-Cas9 approaches to investigate microRNA targeting in human chondrocytes |
| title_sort | evaluation of crispr cas9 approaches to investigate microrna targeting in human chondrocytes |
| work_keys_str_mv | AT chaudhrynm evaluationofcrisprcas9approachestoinvestigatemicrornatargetinginhumanchondrocytes |