Using the CRISPR/Cas9 System to Derive an Isogenic BRCA1 Mutant (187delAG) Strain From a Wild Type BRCA1 Mammary Epithelial Strain
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CitationGreene-Colozzi, April. 2016. Using the CRISPR/Cas9 System to Derive an Isogenic BRCA1 Mutant (187delAG) Strain From a Wild Type BRCA1 Mammary Epithelial Strain. Master's thesis, Harvard Extension School.
AbstractA powerful new genomic engineering technique, the Clustered RegularInterspersed Short Palindromic Repeats (CRISPR), was co-opted from the immunesystem of bacteria and archaea, and uses specific guide RNAs to bring the Cas-9endonuclease to a targeted DNA sequence, which then creates double-stranded breaks inthe genome where a new sequence can be inserted. CRISPR is notable particularly for therelative ease with which one can manipulate the system to generate almost any sequenceof interest, and the high specificity and efficiency for its target (Chu et al., 2015).
The goal of this project was to introduce a well-studied founder mutation (twobase pair deletion), (187delAG; BRCA1mut/+) into BRCA1 wild type (WT; mutationfree;BRCA1+/+) human mammary epithelial cells (HMECs), to generate isogenic WTand BRCA1mut/+ cell strains. Such isogenic strains, with no variability between the wildtype and mutant strain except the mutation of interest, would provide an important toolfor understanding the contribution of BRCA1 mutation towards those early changes inthe cells that lead to breast and ovarian cancer in women with BRCA1 mutations (Silver& Livingston, 2012). This study demonstrated the use of several methods for screeningsingle cell colonies for the small two base pair deletion, which precludes the use ofconventional selection methods. During the process of screening, a reliable strategy wasdeveloped; however, we were not successful in isolating the right clone even afterscreening roughly 350 clones. Nevertheless, this study offers suggestions for futureattempts to introduce mono allelic mutations of the kind described in this study. Amongother things, strategies such as enhancing the high fidelity repair mechanism, blocking the error-prone repair pathway, and employing alternate methods of transfection and cellscreening are strongly recommended for future researchers.
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