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. A., 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+guide+for+functional+analysis+of+BRCA1+variants+of+uncertain+significance.">A guide for functional analysis of BRCA1 variants of uncertain significance.</a> Human Mutation. 33 (11), 1526-1537 (2012).</li><li>Santos, C., 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=Pathogenicity+evaluation+of+BRCA1+and+BRCA2+unclassified+variants+identified+in+Portuguese+breast/ovarian+cancer+families.">Pathogenicity evaluation of BRCA1 and BRCA2 unclassified variants identified in Portuguese breast/ovarian cancer families.</a> Journal of Molecular Diagnostics. 16 (3), 324-334 (2014).</li><li>Starita, L. M., 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+Multiplex+Homology-Directed+DNA+Repair+Assay+Reveals+the+Impact+of+More+Than+1,000+BRCA1+Missense+Substitution+Variants+on+Protein+Function.">A Multiplex Homology-Directed DNA Repair Assay Reveals the Impact of More Than 1,000 BRCA1 Missense Substitution Variants on Protein Function.</a> American Journal of Human Genetics. 103 (4), 498-508 (2018).</li><li>Anantha, R. W., 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=Functional+and+mutational+landscapes+of+BRCA1+for+homology-directed+repair+and+therapy+resistance.">Functional and mutational landscapes of BRCA1 for homology-directed repair and therapy resistance.</a> Elife. 6, (2017).</li><li>Gibson, T. J., Seiler, M., Veitia, R. 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. M., 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=Posttranscriptional+regulation+of+the+breast+cancer+susceptibility+gene+BRCA1+by+the+RNA+binding+protein+HuR.">Posttranscriptional regulation of the breast cancer susceptibility gene BRCA1 by the RNA binding protein HuR.</a> Cancer Research. 68 (22), 9469-9478 (2008).</li><li>Knott1, G. J., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas+guides+the+future+of+genetic+engineering.">CRISPR-Cas guides the future of genetic engineering.</a> Science. 361, 866-869 (2018).</li><li>Sander, J. D., Joung, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas+systems+for+editing,+regulating+and+targeting+genomes.">CRISPR-Cas systems for editing, regulating and targeting genomes.</a> Nature Biotechnology. 32 (4), 347-355 (2014).</li><li>Hess, G. T., 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=Directed+evolution+using+dCas9-targeted+somatic+hypermutation+in+mammalian+cells.">Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells.</a> Nature Methods. 13 (12), 1036-1042 (2016).</li><li>Kim, K., 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=Highly+efficient+RNA-guided+base+editing+in+mouse+embryos.">Highly efficient RNA-guided base editing in mouse embryos.</a> Nature Biotechnology. 35 (5), 435-437 (2017).</li><li>Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., Liu, D. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+overlapping+DNA+fragments.">Enzymatic assembly of overlapping DNA fragments.</a> Methods in Enzymology. 498, 349-361 (2011).</li><li>Nageshwaran, 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=CRISPR+Guide+RNA+Cloning+for+Mammalian+Systems.">CRISPR Guide RNA Cloning for Mammalian Systems.</a> Journal of Visualized Experiments. (140), (2018).</li><li>Findlay, G. M., 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=Accurate+classification+of+BRCA1+variants+with+saturation+genome+editing.">Accurate classification of BRCA1 variants with saturation genome editing.</a> Nature. 562 (7726), 217-222 (2018).</li><li>Kweon, J., Kim, D. E., Jang, A. H., Kim, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas-based+customization+of+pooled+CRISPR+libraries.">CRISPR/Cas-based customization of pooled CRISPR libraries.</a> PLoS One. 13 (6), 0199473 (2018).</li><li>Kim, Y., 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+library+of+TAL+effector+nucleases+spanning+the+human+genome.">A library of TAL effector nucleases spanning the human genome.</a> Nature Biotechnology. 31 (3), 251-258 (2013).</li><li>Sayers, E. W., 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=GenBank.">GenBank.</a> Nucleic Acids Research. 47 (1), 94-99 (2019).</li><li>Hwang, G. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+target+specificity+of+CRISPR+RNA-guided+adenine+base+editors.">Genome-wide target specificity of CRISPR RNA-guided adenine base editors.</a> Nature Biotechnology. 37 (4), 430-435 (2019).</li><li>Clement, K., 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=CRISPResso2+provides+accurate+and+rapid+genome+editing+sequence+analysis.">CRISPResso2 provides accurate and rapid genome editing sequence analysis.</a> Nature Biotechnology. 37 (3), 224-226 (2019).</li><li>Hu, J. 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=Evolved+Cas9+variants+with+broad+PAM+compatibility+and+high+DNA+specificity.">Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.</a> Nature. 556 (7699), 57-63 (2018).</li><li>Nishimasu, 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=Engineered+CRISPR-Cas9+nuclease+with+expanded+targeting+space.">Engineered CRISPR-Cas9 nuclease with expanded targeting space.</a> Science. 361 (6408), 1259-1262 (2018).</li><li>Walton, R. T., Christie, K. A., Whittaker, M. N., Kleinstiver, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unconstrained+genome+targeting+with+near-PAMless+engineered+CRISPR-Cas9+variants.">Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.</a> Science. , (2020).</li><li>Kim, D., 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=Genome-wide+target+specificities+of+CRISPR+RNA-guided+programmable+deaminases.">Genome-wide target specificities of CRISPR RNA-guided programmable deaminases.</a> Nature Biotechnology. 35 (5), 475-480 (2017).</li><li>Zuo, E., 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=Cytosine+base+editor+generates+substantial+off-target+single-nucleotide+variants+in+mouse+embryos.">Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos.</a> Science. 364 (6437), 289-292 (2019).</li><li>Jin, 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=Cytosine,+but+not+adenine,+base+editors+induce+genome-wide+off-target+mutations+in+rice.">Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice.</a> Science. 364 (6437), 292-295 (2019).</li><li>Grunewald, 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=Transcriptome-wide+off-target+RNA+editing+induced+by+CRISPR-guided+DNA+base+editors.">Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.</a> Nature. 569 (7756), 433-437 (2019).</li><li>Doman, J. L., Raguram, A., Newby, G. A., Liu, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+minimization+of+Cas9-independent+off-target+DNA+editing+by+cytosine+base+editors.">Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors.</a> Nature Biotechnology. 38 (5), 620-628 (2020).</li><li>Gaudelli, N. M., 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=Programmable+base+editing+of+A*T+to+G*C+in+genomic+DNA+without+DNA+cleavage.">Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage.</a> Nature. 551 (7681), 464-471 (2017).</li><li>Anzalone, A. V., 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=Search-and-replace+genome+editing+without+double-strand+breaks+or+donor+DNA.">Search-and-replace genome editing without double-strand breaks or donor DNA.</a> Nature. 576 (7785), 149-157 (2019).</li></ol>

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

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Morphology-Based Distinction Between Healthy and Pathological Cells Utilizing Fourier Transforms and Self-Organizing Maps

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 the site of inflammation and are activated to prevent a further spreading of the invasion. This is also reflected by changes in the behavior and the morphological appearance of the immune cells. In cancerous tissue, similar morphokinetic changes have been observed in the behavior of microglial cells: intra-tumoral microglia have less complex 3-dimensional shapes, having less-branched cellular processes, and move more rapidly than those in healthy tissue. The examination of such morphokinetic properties requires complex 3D microscopy techniques, which can be extremely challenging when executed longitudinally. Therefore, the recording of a static 3D shape of a cell is much simpler, because this does not require intravital measurements and can be performed on excised tissue as well. However, it is essential to possess analysis tools that allow the fast and precise description of the 3D shapes and allows the diagnostic classification of healthy and pathogenic tissue samples based solely on static, shape-related information. Here, we present a toolkit that analyzes the discrete Fourier components of the outline of a set of 2D projections of the 3D cell surfaces via Self-Organizing Maps. The application of artificial intelligence methods allows our framework to learn about various cell shapes as it is applied to more and more tissue samples, whilst the workflow remains simple.

Here, we provide a workflow that allows the identification of healthy and pathological cells based on their 3-dimensional shape. We describe the process of using 2D projection outlines based on the 3D surfaces to train a Self-Organizing Map that will provide objective clustering of the investigated cell populations.

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 different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

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 ...

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 basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

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

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 contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

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, whether ovaries contain bacteria has not yet been confirmed yet. Herein, an experiment to detect bacteria in ovarian tissues was introduced. We chose ovarian cancer patients in the cancer group and noncancerous patients in the control group. 16S rRNA gene sequencing was used to differentiate bacteria in ovarian tissues from the cancer and control groups. Furthermore, we predicted the functional composition of the identified bacteria by using BugBase and PICRUSt. This method can also be used in other viscera and tissues since many organs have been proven to harbor bacteria in recent years. The presence of bacteria in viscera and tissues may help scientists evaluate cancerous and normal tissues and may be aid in the treatment of cancer.

Immunohistochemistry staining and 16S ribosomal RNA gene (16S rRNA gene) sequencing were performed in order to discover and distinguish bacteria in cancerous and noncancerous ovarian tissues in situ. The compositional and functional differences of the bacteria were predicted by using BugBase and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt).

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

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice.
Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo.
In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

Culturing CTCs allows a deeper functional characterization of cancer, through assaying specific marker expression, and assessing drug resistance and the ability to colonize the liver among other possibilities. Overall, CTC culture could be a promising clinical tool for personalized medicine to improve patient outcome.

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an ...

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.

Here, we describe a protocol in which an acute lymphoblastic leukemia patient-derived xenograft model is used as a strategy to assess and monitor CD19-targeted chimeric antigen receptor T cell-associated toxicities.

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...