Cloning-free CRISPR
We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each targ...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | en_US |
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Elsevier
2016
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| Online Access: | http://hdl.handle.net/1721.1/100809 https://orcid.org/0000-0003-0521-5855 |
| _version_ | 1826192932550475776 |
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| author | Arbab, Mandana Srinivasan, Sharanya Geijsen, Niels Sherwood, Richard I. Hashimoto, Tatsunori Benjamin |
| author2 | Massachusetts Institute of Technology. Computer Science and Artificial Intelligence Laboratory |
| author_facet | Massachusetts Institute of Technology. Computer Science and Artificial Intelligence Laboratory Arbab, Mandana Srinivasan, Sharanya Geijsen, Niels Sherwood, Richard I. Hashimoto, Tatsunori Benjamin |
| author_sort | Arbab, Mandana |
| collection | MIT |
| description | We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis. |
| first_indexed | 2024-09-23T09:31:22Z |
| format | Article |
| id | mit-1721.1/100809 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T09:31:22Z |
| publishDate | 2016 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | mit-1721.1/1008092022-09-30T15:00:52Z Cloning-free CRISPR Arbab, Mandana Srinivasan, Sharanya Geijsen, Niels Sherwood, Richard I. Hashimoto, Tatsunori Benjamin Massachusetts Institute of Technology. Computer Science and Artificial Intelligence Laboratory Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science Srinivasan, Sharanya Hashimoto, Tatsunori Benjamin We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis. National Institutes of Health (U.S.) (5UL1DE019581) National Institutes of Health (U.S.) (RL1DE019021) National Institutes of Health (U.S.) (1K01DK101684-01) National Institutes of Health (U.S.) (1U01HG007037) National Institutes of Health (U.S.) (5P01NS055923) Harvard Stem Cell Institute (Sternlicht Director's Fund Award) 2016-01-13T16:56:19Z 2016-01-13T16:56:19Z 2015-10 2015-09 Article http://purl.org/eprint/type/JournalArticle 22136711 http://hdl.handle.net/1721.1/100809 Arbab, Mandana, Sharanya Srinivasan, Tatsunori Hashimoto, Niels Geijsen, and Richard I. Sherwood. “Cloning-Free CRISPR.” Stem Cell Reports 5, no. 5 (November 2015): 908–917. https://orcid.org/0000-0003-0521-5855 en_US http://dx.doi.org/10.1016/j.stemcr.2015.09.022 Stem Cell Reports Creative Commons Attribution http://creativecommons.org/licenses/by-nc-nd/4.0/ application/pdf Elsevier Elsevier |
| spellingShingle | Arbab, Mandana Srinivasan, Sharanya Geijsen, Niels Sherwood, Richard I. Hashimoto, Tatsunori Benjamin Cloning-free CRISPR |
| title | Cloning-free CRISPR |
| title_full | Cloning-free CRISPR |
| title_fullStr | Cloning-free CRISPR |
| title_full_unstemmed | Cloning-free CRISPR |
| title_short | Cloning-free CRISPR |
| title_sort | cloning free crispr |
| url | http://hdl.handle.net/1721.1/100809 https://orcid.org/0000-0003-0521-5855 |
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