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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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transience+of+transient+overexpression.">The transience of transient overexpression.</a> Nature Methods. 10 (8), 715-721 (2013).</li><li>Quann, K., Jing, Y., Rigoutsos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-transcriptional+regulation+of+BRCA1+through+its+coding+sequence+by+the+miR-15/107+group+of+miRNAs.">Post-transcriptional regulation of BRCA1 through its coding sequence by the miR-15/107 group of miRNAs.</a> Frontiers in Genetics. 6, 242 (2015).</li><li>Saunus, J. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+editing+of+a+target+base+in+genomic+DNA+without+double-stranded+DNA+cleavage.">Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.</a> Nature. 533 (7603), 420-424 (2016).</li><li>Park, D. S., 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=Targeted+Base+Editing+via+RNA-Guided+Cytidine+Deaminases+in+Xenopus+laevis+Embryos.">Targeted Base Editing via RNA-Guided Cytidine Deaminases in Xenopus laevis Embryos.</a> Molecules and Cells. 40 (11), 823-827 (2017).</li><li>Kweon, J., 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=A+CRISPR-based+base-editing+screen+for+the+functional+assessment+of+BRCA1+variants.">A CRISPR-based base-editing screen for the functional assessment of BRCA1 variants.</a> Oncogene. 39 (1), 30-35 (2020).</li><li>Gibson, D. 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H., 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=Web-based+design+and+analysis+tools+for+CRISPR+base+editing.">Web-based design and analysis tools for CRISPR base editing.</a> BMC Bioinformatics. 19 (1), 542 (2018).</li><li>Kim, D., Kim, D. E., Lee, G., Cho, S. I., Kim, J. 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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 ...

By efficiently inducing point mutations using the CRISPR-mediated cytosine bases editor, the functionality of BRCA1 variants, which is important for disease prevention and diagnosis, can be identified. The advantage of this technique is that it directly mutates endogenously expressed BRCA1, which overcomes the limitation of functional evaluation using exogenously expressed BRCA1. Identification of the loss of function mutations of BRCA1 can be used to predict the chance of developing cancers associated with BRCA1 mutations, such as breast, ovarian, prostate, and pancreatic cancers.It can also be used for finding potential drug targets by searching for essential genes whose viability is reduced through functional depletion. To begin, obtain the BRCA1 genome sequence from GenBank at NCBI and search the 20 base pair target sites with the protospacer adjacent motif sequence, NGGNCCN, around the mutation of interest. The mutation of interest should be located within four to eight nucleotides in the PAM-distal end of the guide RNA target sequences.Order two complimentary oligonucleotides per guide RNA. For the forward oligonucleotides, add CACCG to the 5'end of the guide sequence and for the reverse oligonucleotides, add AAC to the 5'end and C to the 3'end. After receiving the oligonucleotides, resuspend them in distilled water to a final concentration of 100 micromolar.Mix the two complimentary oligonucleotides to a final concentration of 10 micromolar with T4 ligation buffer and heat them to 95 degrees Celsius for five minutes then cool them to room temperature for annealing. Digest pRG2 using the restriction enzyme Bsa1 for one hour at 37 degrees Celsius, then run the digested product on a 1%agarose gel and purify the appropriate sized band. Ligate the annealed oligonucleotide duplex to the vector DNA using the purchased DNA ligase according to the manufacturer's protocol and transform them into DH5-alpha competent cells.Add the transformants to an agar plate containing 100 micrograms per milliliter ampicillin and incubate the plate overnight at 37 degrees Celsius. Purify plasma DNA from several transformants and analyze their guide RNA sequences by Sanger sequencing with primers that prime at the U6 promoter. Seed five times 10 to the fifth HAP1-BE3 cells per well in 24-well plates one day prior to transfection and culture them to reach 70 to 80%confluence for transfection.Transfect BRCA1 targeting guide RNAs using the purchased transfection reagents according to the manufacturer's protocol. Use one microgram of BRCA1 targeting guide RNAs to induce CG to TA conversion at BRCA1 target sites then incubate the cells at 37 degrees Celsius and subculture every three to four days. Harvest the cell pellets three, 10 and 24 days after transfection to analyze base editing efficiencies.Extract genomic DNA using the genomic DNA purification kit. Design the first PCR primers to amplify BRCA1 target sites. Although there is no restriction on the size of the first PCR product, a product size of less than one kilobases is recommended to efficiently amplify a specific region.Design the second PCR primers located inside the first PCR product, taking into consideration the size of the amplicon according to the NGS read length. In order to attach essential sequences for NGS analysis, add additional sequences to the 5'ends of the second PCR primers. Amplify the BRCA1 target sites on the genomic DNA obtained from the three time points using high-fidelity polymerase.For the first PCR reaction, use 100 nanograms of genomic DNA for amplification over 15 cycles. For the second PCR reaction, use one microliter of the first PCR product for amplification over 20 cycles. Run five microliters of the second PCR product on 2%agarose gel and confirm the size.Then attach the essential sequences for NGS analysis by amplifying the second PCR product using the primers listed in the text manuscript. Amplify each sample using different primer sets. Use one microliter of the second PCR product for up to 30 cycles of amplification with a high-fidelity polymerase.Run five microliters of the PCR product on 2%agarose gel to confirm the size and purify the amplicon using a commercial PCR cleanup kit. Mix each sample in equal amounts to create an NGS library. Quantify the NGS library using a spectrophotometer and dilute it to a concentration of one nanomolar using resuspension buffer or 10 millimolar Tris-HCl at pH 8.5.Prepare 100 microliters of the library diluted to the appropriate loading concentration. As a control combined PhiX with the diluted sample. Load the library onto the cartridge and run NGS according to the manufacturer's protocol.Obtain over 10, 000 reads per target amplicon for in-depth analysis of base editing efficiency. This protocol was used to perform a functional assessment of endogenous BRCA1 variants generated by CRISPR-based cytosine base editors. CAS-9 and guide RNAs were transfected into HAP1 cell lines to disrupt BRCA1, and mutation frequencies were analyzed.Mutation frequencies decreased significantly over time in HAP1 cell lines, indicating that BRCA1 is an essential gene for cell viability in these cells. To investigate whether CG to TA substituted variants affect the function of BRCA1, the DNA plasmids and coding guide RNAs that could induce each mutation were transfected to have HAP1-BE3 cell lines. And the substitution frequencies were analyzed.The relative substitution frequencies of 3598C to T, a pathogenic variant that induces a nonsense mutation, dramatically decreased. Whereas those of 4527C to T, a benign variant that induces a nonsense mutation, remained similar with time. It was found that nucleotide substitution frequencies of these three variants decreased in a time-dependent manner.Since it is necessary to obtain a high initial mutation frequency in order to clearly recognize the change in cell viability with the passing of the date, it is a priority to establish optimized transfection conditions. This procedure can be applied for verifying other unknown genetic variants such as BRCA VUS. It may provide clues to the pathogenicity of patient-derived unknown variants and their treatment.

functional-assessment-brca1-variants-using-crispr-mediated-base

Research

JoVE Journal

Cancer Research

5.3K Aufrufe.  University of Ulsan College of Medicine. Durch die effiziente Induktion von Punktmutationen mit dem CRISPR-vermittelten Cytosin-Basis-Editor kann die Funktionalität von BRCA1-Varianten, die für die Prävention und Diagnose von Krankheiten wichtig ist, identifiziert werden. Der Vorteil dieser Technik ist, dass sie direkt endogen ausgedrücktes BRCA1 mutiert, was die Einschränkung der funktionellen Bewertung mit exogen exogen exogen ausgedrücktem BRCA1 überwindet. Die Identifizierung des Verlusts von Funktionsmutationen von BRCA1...