Year: 2020
Type: article
Abstract: 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.
Source: Genes
Institution: The University of Texas MD Anderson Cancer Center
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,901
Awards: Molecular Regulation of the Perinatal Male Germ Cell Niche, Instituting innovative CRISPR/Cas9 technology at an NCI Comprehensive Cancer Center transgenic facility, Role of BHC80 in spermatogenesis, A Novel Role for MAGI1 in regulating non-canonical LATS signaling and atherosclerotic plaque formation, G6134912941, Pathological flow-induced endothelial damage and plaque erosion, Cancer Center Support Grant
OpenAlex ID: W3033467537