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Rapid Evaluation of CRISPR Guides and Donors for Engineering Mice

Genes (Basel). 2020-06-01; 
Elena McBeath, Jan Parker-Thornburg, Yuka Fujii, Neeraj Aryal, Chad Smith, Marie-Claude Hofmann, Jun-Ichi Abe, Keigi Fujiwara
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Codon Optimization … Using GenScript's mouse codon frequency tables (GenScript Codon Usage Frequency Table Tool, https://www.genscript.com/tools/codon-frequency-table), the codon used for the desired amino acid change was selected to have an about equal or slightly higher frequency tRNA … Get A Quote

摘要

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, ... More

關鍵詞

CRISPR/Cas9, blastocyst, gene targeting, genetic engineering, silent mutation
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