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 in pCMV-BE3 (Table of Materials) by PCR using high-fidelity polymerase. Design the PCR primer to contain overlapping sequences of the digested vector (from step 1.1.2) for isothermal assembly as follows17: 
		primer-F: 5&#8217;-TTTGCCGCCAGAACACAGGACCGGTTCTAGA GCCGCCACCATGAGCTCAGAG-3&#8217;, 
		primer-R: 5&#8217;-CAGAGAGAAGTTTGTTGCGCCGGATCC GACTTTCCTCTTCTTCTTGGG-3&#8217; 
		NOTE: Nucleotides shown in underlined are overlapped with the digested vector and these sequences will be specific to the destination vector chosen by the user.</li>
		<li>Digest lentiCas9-blast (Table of Materials) plasmid DNA with the restriction enzymes, XbaI and BamHI (1 unit per 1 &#956;g) for 1 h at 37 &#176;C. Run the digested product on a 0.8% agarose gel and purify the appropriate-sized bands (8.6 kb) using a commercial gel extraction kit (Table of Materials).</li>
		<li>Clone the amplified BE3 PCR product and digested lentiCas9-Blast vector using the isothermal assembly kit (Table of Materials). Use a total of 0.02-0.5 pmol of DNA fragments and a 1:3 ratio of vector:insert. Transform the assembly product into DH5 alpha-competent cells18. Add the transformants on an agar plate containing ampicillin (100 &#181;g/mL) and incubate the plate overnight at 37 &#176;C.</li>
		<li>Pick several colonies and inoculate them in 4 mL of LB (lysogeny broth) medium containing ampicillin (100 &#956;g/mL) and grow the culture in a shaking incubator at 180 rpm.</li>
		<li>Purify plasmid DNA using a commercial plasmid DNA purification kit (Table of Materials) according to the manufacturer&#8217;s protocols.</li>
		<li>To confirm DNA cloning success, analyze the BE3 sequences of each purified plasmid DNA (from step 1.1.5) by Sanger sequencing using standard and BE3-specific primers (Supplementary Table 1) and select the exact cloned lentiBE3-blast construct. 
		NOTE: To analyze the results of Sanger sequencing, we recommended several tools such as BLAST (https://blast.ncbi.nlm.nih.gov/) and CLUSTALW (https://www.genome.jp/tools-bin/clustalw).</li>
	</ol>
	</li>
	<li>Cell culture and generation of HAP1-BE3 cell lines
	<ol>
		<li>Maintain cells in a healthy condition and in an actively dividing state. Culture HAP1 cells in Iscove&#8217;s modified Dulbecco&#8217;s medium (Table of Materials) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Culture HEK293T/17 cells in Dulbecco&#8217;s modified Eagle&#8217;s medium (Table of Materials) containing 10% FBS and 1% penicillin/streptomycin. 
		NOTE: HAP1 cell is useful for genetic research because of it is nearly haploid cells. However, the cells can spontaneously return to a diploid state in cell culture. Enrichment Hoechst 34580 stained 1n-population using flowcytometry will be helpful for maintenance of haploidy of HAP1 cells19.</li>
		<li>Seed 5 x 106 HEK293T/17 cells on a 100 nm dish 1 day before transfection. On the day of transfection, transfect the plasmid DNA (15 &#181;g of lentiBE3-blast, 9 &#181;g of psPAX2 for viral packaging, and 6 &#181;g of pMD2.G for vial envelope of 2nd generation lentiviral packaging system) using commercial transfection reagents according to the manufacturer&#8217;s protocols (Table of Materials)20. Change the medium at 6 h post-transfection and harvest virus-containing medium at 48 h or 72 h post-transfection. Filter the supernatant using a 0.45 &#181;m filter.</li>
		<li>Transduce the lentiviral particles of BE3 into HAP1 cells at a MOI (multiplicity of infection) of 0.1. To adjust for an appropriate MOI, use serially diluted concentrations of virus. Remove the medium and replace it with adjusted viral supernatant and fresh culture medium.</li>
		<li>One day after transduction, change the medium to 10 &#181;g/mL of blasticidin (Table of Materials)-containing medium and select the transduced cells for 3 days with blasticidin. After blasticidin selection, select a well with ~10% of surviving cells for the next step.</li>
		<li>After blasticidin selection, seed the transduced cells on 96-well plates at a density of 0.5 cells/well in order to isolate single clones (e.g., dilute 50 cells in 20 mL (0.5 cells per 200 &#181;L) culture medium and aliquot 200 &#181;L per well in 96 well plate). Incubate the 96-well plates for 2 weeks and pick the single colonies to confirm the BE3 activity.</li>
		<li>Divide the single colonies into two sets, one sets for testing BE3 activity and other for maintenance. Transduce the lentiviral particles of gRNAs into one of the sets to confirm the BE3 activity. Analyze the mutation frequency of each colony using T7 endonuclease I (T7E1, Table of Materials) assay or by performing the targeted deep sequencing technique (step 4)21. Select appropriate single clones that are healthy and have a highly active BE3. 
		NOTE: To validate exact mutation frequencies, we recommend the targeted deep sequencing than T7E1 assay. HEK2 (5&#8217;-GAACACAAAGCATAGACTGCGGG-&#8216;3) is well validated target site for BE3.</li>
	</ol>
	</li>
</ol>
2. Design and construction of BRCA1 targeting gRNAs
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61557/61557fig02.jpg" /> 
Figure 2: An example of a gRNA plasmid DNA.&#160;(A) To effectively edit the target sequence with BE3, an NGG PAM (CCN PAM) that places the target C (target G) within a five-nucleotide window is required. NGG PAM is shown in red and the base editing window is represented by a gray box. (B) The gRNA sequence for c.8047C&#62;T (H1283Y) base editing is indicated and the target C:G pairs are shown in red while the active window is highlighted by a gray box. For gRNA cloning, the overhang sequences indicated in bold are added at both the 5&#697; ends. Templates for gRNA were generated by annealing the two complementary oligonucleotides. <a href="https://www.jove.com/files/ftp_upload/61557/61557fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
NOTE: Positive and negative controls of BRCA1 variants are essential. In this study, c.5252G&#62;A (R1751Q) and c.4527C&#62;T (Y1509Y) are used as benign controls. c.191G&#62;A (C64Y), 81-1G&#62;A, and c.3598C&#62;T (Q1200*) are used as pathogenic controls. Target sequences of each gRNA are listed in Supplementary Table 1.
<ol>
	<li>Obtain the BRCA1 genome sequence from GenBank at NCBI22.</li>
	<li>Search the 20-bp target sites with the Protospacer Adjacent Motif (PAM) sequence &#8220;NGG&#8221; and &#8220;CCN&#8221; around mutation of interest. The mutation of interest should be located in 4&#8211;8 nucleotides in the PAM-distal end of the gRNA target sequences because of the active window of BE3 is 4&#8211;8 nucleotides in the PAM-distal end of the gRNA target sequences14. In the case of c.8047C&#62;T (H1283Y)- and c.5252G&#62;A (R1751Q)-targeting gRNAs, 5&#697;-TCAGGAACATCACCTTAGTG-AGG-3&#697; and 5&#697;-CCA-CCAAGGTCCAAAGCGAGCAA-3&#697;, the sequences in bold are active windows. C to T and G to A conversions occur with NGG and CCN PAM, respectively (Figure 2A). 
	NOTE: BE-Designer (http://www.rgenome.net/be-designer/)23 is useful web-based tools for gRNA design.</li>
	<li>Order two complementary oligonucleotides per gRNA; for the forward oligonucleotides, add &#8220;CACCG&#8221; to the 5&#697; end of the guide sequence, and for the reverse oligonucleotides, add &#8220;AAAC&#8221; to the 5&#8217; end and &#8220;C&#8221; to the 3&#8217; end. These additional sequences are specific to the destination gRNA expression vector (from step 2.5) used for this protocol, and users should be adjusted for alternative gRNA expression vectors (Figure 2B).</li>
	<li>Resuspend the oligonucleotide in distilled water at a final concentration of 100 &#181;M. Mix the two complementary oligonucleotides to a final concentration of 10 &#181;M with T4 Ligation buffer (Table of Materials) and heat them at 95 &#176;C for 5 min and cool them at room temperature for annealing.</li>
	<li>Digest pRG2 using the restriction enzyme, BsaI (1 unit per 1 &#956;g) for 1 h at 37 &#176;C (Table of Materials). Run the digested product on 1% agarose gel and purify the appropriate-sized band (2.5 kb). 
	NOTE: Instead of using a classic digestion and ligation method, other cloning method such as Golden Gate cloning can be used.</li>
	<li>Ligate the annealed oligonucleotide duplex to the vector DNA using the purchased DNA ligase (Table of Materials) according to the manufacturer&#8217;s protocol and transform them into DH5 alpha-competent cells (Other E. coli strains which widely used for subcloning also can be used).</li>
	<li>Add the transformants to an agar plate containing ampicillin (100 &#956;g/mL) and incubate the plate overnight at 37 &#176;C. Purify plasmid DNA from several transformants and analyze their gRNA sequences by Sanger sequencing using primers which prime at U6 promoter (Table of Materials).</li>
</ol>
3. Creation of BRCA1 variants using CRISPR-mediated base editing tools
NOTE: If HAP1-BE3 cell lines is not used, BE3-encoding plasmid DNA can be co-transfected with BRCA1-targeting gRNA. Compared to co-transfection of BE3 and gRNA plasmids, transfection of gRNA plasmid to HAP-BE3 cells induce efficient base editing up to 3-fold at target locus in our hands.
<ol>
	<li>Seed 5 x 105 HAP1-BE3 cells (or HAP1 cells in case of co-transfection methods) per well in 24-well plates 1 day prior to transfection. At the time of transfection, culture cells to reach an appropriate density (70%&#8211;80% confluence).</li>
	<li>Transfect BRCA1-targeting gRNAs using the purchased transfection reagents (Table of Materials) according to the manufacturer&#8217;s protocol. Use 1 &#181;g of BRCA1-targeting gRNAs (with 1 &#181;g of BE3-encoding plasmid DNA in case of co-transfection methods) to induce C:G to T:A conversion at BRCA1 target sites. Incubate the cells at 37 &#176;C and subculture every 3&#8211;4 days.</li>
	<li>Harvest the cell pellets 3, 10, and 24 days after transfection to analyze base editing efficiencies (Samples from 3 days after transfection are analyzed regrading as day 0 samples).</li>
	<li>Extract genomic DNA using the genomic DNA purification kit (Table of Materials). 
	NOTE: We recommend optimizing transfection conditions with variable ratio of reagent to DNA. Optimal condition for HAP1 transfection is 4:1 ration of reagent to DNA in our hands.</li>
</ol>
4. Sample preparation for Illumina next-generation sequencing (NGS)
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61557/61557fig03.jpg" /> 
Figure 3: Preparation for next-generation sequencing.&#160;The 1st PCR primer was designed to amplify the BRCA1 target site on genomic DNA. The 2nd PCR primer was designed such that its sequences are located more inside than the 1st PCR primer sequences. Additional sequences shown as a yellow bar were added at both ends of the 2nd PCR primer to attach the essential sequences for performing next-generation sequencing. <a href="https://www.jove.com/files/ftp_upload/61557/61557fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Design the 1st PCR primers to amplify BRCA1 target sites. Although there is no restriction on the size of the 1st PCR product, a product size of &#60;1 kb is recommended to efficiently amplify a specific region (Figure 3).</li>
	<li>Design the 2nd PCR primers located inside the 1st PCR product. Consider the size of the amplicon according to the NGS read length (For example, the size of the amplicon product should be smaller than 300 bp for 2 &#215; 150 bp paired-end run to merge each reads). In order to attach essential sequences for NGS analysis, add additional sequences to the 5&#8217; end of the 2nd PCR primers as follows: for the forward primers, add 5&#8217;-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3&#8217; sequences to the 5&#8217; end, and for the reverse primers, add 5&#8217;-GTGACTGGAGTTCAGACGTGTGCTCTTCCG ATCT-3&#8217; sequences to the 5&#8217; end. 
	NOTE: We used nested PCR to reduce the amplicon of non-specific binding by preventing primers from attaching to non-target sequence.</li>
	<li>Amplify the BRCA1 target sites on the genomic DNA obtained from three time points. Use high-fidelity polymerase according to the manufacturer&#8217;s protocol for minimizing PCR errors. For the 1st PCR reaction, use 100 ng of genomic DNA for amplification over 15 cycles. For the 2nd PCR reaction, use 1 &#181;L of the 1st PCR product for amplification over 20 cycles. Run 5 &#181;L of the 2nd PCR product on 2% agarose gel and confirm the size.</li>
	<li>In order to attach the essential sequences for NGS analysis, amplify the 2nd PCR product using the primers listed below. For the PCR reaction, 1 &#181;L of the 2nd PCR product is used for amplification up to 30 cycles using high-fidelity polymerase.
	<ol>
		<li>To perform multiplex sequencing for large numbers of libraries to be pooled and sequenced simultaneously, use forward (D501 &#8211; D508) and reverse (D701 &#8211; D712) primers, which have unique index sequence (Supplementary Table 1). Amplify each sample using different primers sets to conduct the unique dual indexing strategy.</li>
		<li>Run 5 &#181;L of the PCR product on 2% agarose gel to confirm the size, and purify the amplicon using a commercial PCR clean-up kit (Table of Materials). Mix each sample in equal amounts to create an NGS library.</li>
	</ol>
	</li>
	<li>Quantify the NGS library at 260 nm wavelength using spectrophotometers and dilute the NGS library to concentration of 1 nM using resuspension buffer or 10 mM Tris-HCl, pH 8.5. Prepare 100 &#181;L of the library diluted to the appropriate loading concentration depending on the library types (For example, prepare 200 pM of the library for Nextera DNA Flex).
	<ol>
		<li>As a control, combine phiX with the diluted sample appropriate for the type of kit.</li>
		<li>Load the library onto the cartridge and run NGS according to the manufacturer&#8217;s protocol. Illumina iSeq 100 or Miseq systems can sequence the amplicons of various length up to 300 bp for single-read or paired end. We recommended over 10,000 reads per target amplicon for in-depth analysis of base editing efficiency.</li>
	</ol>
	</li>
</ol>
5. Analysis of base editing efficiency for the functional assessment of BRCA1 variants
<ol>
	<li>Analyze the base editing efficiencies using MAUND24. The base editing efficiency is calculated as described below. 
	<img alt="Equation 1" src="/files/ftp_upload/61557/61557equ01.jpg" /> 
	If multiple cytosines are present in BE3 active window, only the C to T conversion that is targeted for evaluating the BRCA1 variant is considered. For example, if the sequences of BE3 active window are &#8220;C4A5T6C7T8,&#8221; the possible sequences generated by base editing are &#8220;T4A5T6C7T8&#8221;, &#8220;C4A5T6T7T8&#8221;, and &#8220;T4A5T6T7T8&#8221;. At this time, if position 4 is the targeted position for the desired BRCA1 variant, then only the &#8220;&#8220;T4A5T6C7T8&#8221; sequence is considered for calculating base editing efficiency. 
	NOTE: We also recommend other web-tools for analysis of base editing activity such as CRISPResso225, and BE-analyzer23.</li>
	<li>Verify the results using positive and negative controls of BRCA1 variants. The base editing efficiencies of the benign control should remain the same, whereas those of the pathogenic control should decrease over time.</li>
	<li>Calculate the relative base editing efficiency of BRCA1 variants and determine their pathogenicity. The significant differences between Day 0 and Day 21 samples are analyzed appropriate statistical analysis such as t-test.</li>
</ol>

<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>

The authors have nothing to disclose.

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 ...

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

Research

JoVE Journal

Cancer Research

5.3K Views.  University of Ulsan College of Medicine. 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.

Video: Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

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 ...

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) nitrosamine (BBN) carcinogen are advantageous over cell line-based models because they closely replicate the genomic profiles of human tumors, and, unlike cell models and xenografts, they provide a good opportunity for the study of immunotherapies. However, bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Described here is a BBN mouse model and protocol to evaluate bladder cancer tumor burden in vivo using a fast and reliable magnetic resonance (MR) sequence (true FISP). This method is simple and reliable because, unlike ultrasound, MR is operator-independent and allows for the straightforward post-acquisition image processing and review. Using axial images of the bladder, analysis of regions of interest along the bladder wall and tumor allow for the calculation of bladder wall and tumor area. This measurement correlates with ex vivo bladder weight (rs= 0.37, p = 0.009) and tumor stage (p = 0.0003). In conclusion, BBN generates heterogeneous tumors that are ideal for evaluation of immunotherapies, and MRI can quickly and reliably assess tumor burden prior to randomization to experimental treatment arms.

Murine bladder tumors are induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen (BBN). Bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Here we present a fast, reliable MRI protocol to assess tumor size and stage.

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 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 ...