Name: functional assessment of brca1 variants using crispr-mediated base editors
Uploaded: 2021-02-28T13:00:00
Description: Watch this Scientific Journal Video about Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors at JoVE.com

This work was supported by the National Research Foundation of Korea (grants 2017M3A9B4062419, 2019R1F1A1057637, and 2018R1A5A2020732 to Y.K.).

NOTE: Method 1 (generation of HAP1-BE3 cell lines) is optional. Instead of constructing a BE3-expressing cell line, BE3-encoding plasmid DNA can be co-transfected with gRNA-encoding plasmid DNA. Other variants of cytosine base editors, such as BE4max, also can be used for highly efficient base editing.
1. Generation of HAP1-BE3 cell lines
<ol>
	<li>Construction of plasmid DNA
	<ol>
		<li>To construct the lentiBE3-blast plasmid DNA for lentivirus production, amplify the BE3 coding sequences i...

<ol>
	<li>Roy, R., Chun, J., Powell, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1+and+BRCA2:+different+roles+in+a+common+pathway+of+genome+protection.">BRCA1 and BRCA2: different roles in a common pathway of genome protection.</a> Nature Reviews Cancer. 12 (1), 68-78 (2011).</li><li>Kuchenbaecker, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risks+of+Breast,+Ovarian,+and+Contralateral+Breast+Cancer+for+BRCA1+and+BRCA2+Mutation+Carriers.">Risks of Breast, Ovarian, and Contralateral Breast Cancer for BRCA1 and BRCA2 Mutation Carriers.</a> Journal of the American Medical Association. 317 (23), 2402-2416 (2017).</li><li>Millot, G. 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This protocol describes a simple method for functional assessments of BRCA1 variants using CRISPR-meditated cytosine base editor. The protocol describes methods for the design of gRNAs at target locus and construction of the plasmid DNAs from which they are expressed. Cytosine base editors induce nucleotide conversion in an active window (in case of BE3, nucleotides 4&#8211;8 in the PAM-distal end of the gRNA target sequences). The researcher should carefully choose target sequences because all cytosines in acti...

The breast cancer type 1 susceptibility gene (BRCA1) is a widely known tumor suppressor gene. Because the BRCA1 gene is related to the repair of DNA damage, mutations in this gene would lead to a greater risk of cancer development in an individual1. Breast, ovarian, prostate, and pancreatic cancers are linked to inherited loss-of-function (LOF) mutations of the BRCA1 gene2. Functional assessment and identification of BRCA1 variants may help in preventing and diagnosing the various diseases. To address function of BRCA1 variants, several methods have been developed and broadly used for investigating the pathogenicity of BRCA1 variants such as embryonic stem cell viability assays, fluorescent reporter assays, and therapeutic drug-based sensitivity assays3,4,5,6. Although these methods have assessed the function of a lot of BRCA1 variants, the methods involving exogenously expressed BRCA1 variants pose limitations in terms of overexpression that might affecting downstream regulation, gene dosage, and protein folding7. Furthermore, these assays cannot be harnessed to the posttranscriptional regulation such as mRNA splicing, transcript stability, and effect of untranslated region8,9.
CRISPR-Cas9 system enables targeted genome editing in living cells and organisms10. Through a single-guide RNA, Cas9 can induce double-strand breaks (DSBs) in chromosomal DNA at specific genomic loci in order to activate two DNA repair pathways: error-prone nonhomologous end-joining (NHEJ) pathway and error-free homology-directed repair (HDR) pathway11. HDR is a precise repair mechanism; however, DSBs induced by Cas9 nuclease for HDR often results in unwanted insertion and deletion (indel) mutation. Additionally, it needs homologous donor DNA templates for repairing DNA damage and has relatively low efficiency. Recently, Cas9 nickase (nCas9) have been fused with cytidine deaminase domains for targeting C:G to T:A substitutions, without the need for homologous DNA templates and DNA double strand breaks12,13,14,15. Using the cytosine base editor, we developed a new method for functional analysis of BRCA1 variants16.
In this study, we used CRISPR-mediated cytosine base editor, BE314, which induces efficient C:G to T:A point mutations, for implementing the functional assessment of BRCA1 variants and successfully identified the functions of several BRCA1 variants (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61557/61557fig01.jpg" /> 
Figure 1: An overview of the workflow for functional assessment.&#160;(A) Schematic showing the functional assessment of BRCA1. Because the LOF of BRCA1 affects cell viability, when the BRCA1 mutation is pathogenic, the cells die as the passage number increases. (B) Stages of the functional assessment of BRCA1. Dotted box is optional. It can be replaced by co-transfection of gRNA expressing and BE3 expressing plasmids DNA. <a href="https://www.jove.com/files/ftp_upload/61557/61557fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>BamHI</td>
			<td>NEB</td>
			<td>R3136</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1113903</td>
			<td>Drug for selecting transduced cells</td>
		</tr>
		<tr>
			<td>BsaI</td>
			<td>NEB</td>
			<td>R0535</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>DNeasy Blood &#38; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td>Genomic DNA prep. kit</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td>Medium for HEK293T/17 cells</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>16000036</td>
			<td>Supplemetal for cell culture</td>
		</tr>
		<tr>
			<td>FuGENE HD Transfection Reagent</td>
			<td>Promega</td>
			<td>E2311</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Gibson Assembly Master Mix</td>
			<td>NEB</td>
			<td>E2611L</td>
			<td>Gibson assembly kit</td>
		</tr>
		<tr>
			<td>Iscove&#8217;s modified Dulbecco&#8217;s medium</td>
			<td>Gibco</td>
			<td>12440046</td>
			<td>Medium for HAP1 cells</td>
		</tr>
		<tr>
			<td>lentiCas9-Blast</td>
			<td>Addgene</td>
			<td>52962</td>
			<td>Plasmids DNA for lentiBE3 cloning</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher Scientific</td>
			<td>11668027</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td>Transfection materials</td>
		</tr>
		<tr>
			<td>pCMV-BE3</td>
			<td>Addgene</td>
			<td>73021</td>
			<td>Plasmids DNA for lentiBE3 cloning</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td>Supplemetal for cell culture</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530SQ</td>
			<td>High-fidelity polymerase</td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td>Plasmids DNA for virus prep.</td>
		</tr>
		<tr>
			<td>pRG2</td>
			<td>Addgene</td>
			<td>104174</td>
			<td>gRNA cloning vector</td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>Addgene</td>
			<td>12260</td>
			<td>Plasmids DNA for virus prep.</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>Plasmid DNA prep. Kit</td>
		</tr>
		<tr>
			<td>QIAquick Gel extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>Gel extraction kit</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>PCR product prep. kit</td>
		</tr>
		<tr>
			<td>Quick Ligation Kit</td>
			<td>NEB</td>
			<td>M2200</td>
			<td>Ligase for gRNA cloning</td>
		</tr>
		<tr>
			<td>T7 Endonuclease I</td>
			<td>NEB</td>
			<td>M0302</td>
			<td>Materials for T7E1 assay</td>
		</tr>
		<tr>
			<td>XbaI</td>
			<td>NEB</td>
			<td>R0145</td>
			<td>Restriction enzyme</td>
		</tr>
	</tbody>
</table>

functional assessment of brca1 variants using crispr-mediated base editors

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C&#62;T, c.154C&#62;T, c.3847C&#62;T, c.5056C&#62;T, and c.4986+5G&#62;A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.

The experimental approaches described in this protocol enable the functional assessment of endogenous BRCA1 variants generated by CRISPR-based cytosine base editors. To select appropriate cell lines for the functional assessment of BRCA1 variants, researchers should confirm that BRCA1 is essential gene in the targeted cell lines. For example, we first transfected Cas9 and gRNAs into HAP1 cell lines to disrupt BRCA1 and analyzed mutation frequencies by targeted deep sequencing. We found...

Watch this Scientific Journal Video about Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors at JoVE.com

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

People with BRCA1 mutations have a higher risk of developing cancer, which warrants accurate evaluation of the function of BRCA1 variants. Herein, we described a protocol for functional assessment of BRCA1 variants using CRISPR-mediated cytosine base editors that enable targeted C:G to T:A conversion in living cells.

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter ...

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The appearance and the movements of immune cells are driven by their environment. As a reaction to a pathogen invasion, the immune cells are recruited to ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Characterization and Functional Prediction of Bacteria in Ovarian Tissues

The theory of a "sterile" female upper reproductive tract has been encountering increasing opposition due to advancements in bacterial detection. However, ...