Rapid Evaluation of CRISPR Guides and Donors for Engineering Mice
| Autor: | Jan Parker-Thornburg, Elena McBeath, Yuka Fujii, Neeraj K. Aryal, Chad Smith, Keigi Fujiwara, Marie Claude Hofmann, Jun Ichi Abe |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Male
0301 basic medicine Silent mutation lcsh:QH426-470 Biology Article gene targeting Animals Genetically Modified Mice 03 medical and health sciences chemistry.chemical_compound symbols.namesake 0302 clinical medicine Genetics Animals CRISPR Guide RNA CRISPR/Cas9 Genetics (clinical) Mice Knockout Sanger sequencing genetic engineering silent mutation Cas9 blastocyst Point mutation Molecular biology genomic DNA lcsh:Genetics 030104 developmental biology chemistry Mutation symbols CRISPR-Cas Systems 030217 neurology & neurosurgery DNA RNA Guide Kinetoplastida |
| Zdroj: | Genes Volume 11 Issue 6 Genes, Vol 11, Iss 628, p 628 (2020) |
| ISSN: | 2073-4425 |
| DOI: | 10.3390/genes11060628 |
| Popis: | Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut. |
| Databáze: | OpenAIRE |
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