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Classification of BRCA1 Variants with Saturation Genome Editing
Authors: Findlay GM, Daza RM, Martin B, Zhang MD, Leith AP, Gasperini M, Janizek JD, Huang X, Starita LM, Shendure J. 2018. Accurate classification of BRCA1 variants with saturation genome editing. Nature. 562(7726): 217-222.
Introduction
The advancement in technology has enhanced gene sequencing, thereby shedding light on the role of genetically inherited mutations. The rare, pathogenic diseases and dual functional disorders caused molecule characterization and derived gene information.
The testing for Cancer risk assessment has been of high interest for many scientists. Many clinicians and geneticists started to be concerned about the genetic variant found in cancer patients and the relatable people (Federici and Soddu 2020).
Variants of uncertain significance (VUS) have been found to limit the genetic information’s clinical utility fundamentally. BRCA1 neutralizes the impact they cause in the genetic information, a tumor moderator in which the sprouts functionally lose the variant. Therefore, most develop cancer of the ovary and the breast. In this case, a saturation of the genome editing assay of 96.5% of all possible single nucleotide variants (SNVs) was employed with 13 exons.
This article summarizes the findings, methods, evaluation, and utility of the results and their possible applications from this respect.
Q1. Summary of the findings
Almost all the genes in the HDR pathway of BRCA1, BRCA2, PALB2, and BARD1 in the hereditary cancer were predisposed and critical in the human haploid cell line HAP1. The change was confirmed by transferring the cells of plasmodia HAP1 cells with Cas9 and controlled by RNAs, aiming to target each of the genes. Besides, the was experienced high death of the cells within the light of microscopy and the luminescence-based survival assay of a gene that substantially reduced the HAP1 cells’ viability.
Conversely, this technology’s most essential advent is introducing the NGS cancer investigation that created the paradoxical shortage of the answers to face and a massive quantity of information from the high-throughput technology (Morash et al. 2018).
Similarly, it was found that cell death is consequent to the targeted sequence of the BRACA1 genes mutated and expressed a widespread selection against the frameshifting of the indels. Thus, these findings overall give the necessity for the HDR pathways of the cells.
Methods 
HDR Pathway Essential Analysis of HAP1 cells
Here, through induced pluripotent stem cell factors to the KBM7 cells, the HAP1 cells are essentially derived. The HAP1 gene was essentially obtained from the resulted scores and filtered with the genes’ insertions more than 20 mapped in the gene trap insertions (N=14306). Additionally, the Gene Ontology (GO) was recombined with the homologous 78 HDR genes, where 66 were taken from the total 14306 genes and used in the analysis. The genes’ ranking was done by an ordered value of (q-value) representing the low to high, and the other proportion was a trap in the gene orientation.

1. gRNA design and cloning

The cloning of gRNAs used in the SGE critically experimented with PX459 and all the CRISPR. This resulted in a plasmid expressing the gRNA from the U6 promoter with Cas9-2A puromycin included.  Later, the targeted Cas9 was chosen from the SGE, and an experiment was carried out in the following criteria. They were inducing cleavage within BARCA1 through a coding sequence, targeting the genomic site permissive to a more synonymous. Induction within the guanine dinucleotide of the protospacer or the PAM, and finally having a targeted maximal and minimal predicted off the activity.

1. HAP1 Cell Culture

This method comprised the use of standardized dominance of the WT HAP1 cells for example the Haplogens and the Horizon. The modified Medium of Iscove Dulbecco’s and L-glutamine, and 25Mm HEPES, are required to supplement 10 percent of fetal serum. Similarly, the method requires 1 percent of the penicillin-streptomycin (GIBCO). A plate with a temperature of 37C is set with a 5% circulation of CO2. Before it becomes confluent, the air is passed and so that the cells are washed once. Therefore, to sort out the affected population of cells before transfection, DNA is washed with Hoechst 34580(BD Biosciences) at 5ug/ml for approximately one hour at a temperature of about 37C. The FACS is carried out to separate 1-2×106 cells from the Hoechst peak low intensity.
Evaluation of the Study 
The study forms one of the most important clinical studies target to help over 2500 variants of unknown significance for the BRCA1 gene. From the recent publications, the paper records deleterious BRACA1 variants. This technique is crucial in modifying the BRCA1 gene in HAP1 cells, the cells dependent on the functional BRCA1 gene for growth, which helps the mutations and the assessment of which are not applicable (Adamovich 2019).
The Utility of Findings and Possible Applications 
Ataxia –Telangiectasia, the ATM gene, is one significant neoplastic utility have found in the paper. The ATM gene is a rare autosomal receptor of genetic diseases generally caused by biallelic mutations. It is determined at the early stages of the disorder, marked by the cerebellar ataxia, immunodeficiency, and predisposition to cancer. ATM gene is a VUS utility applied in the Mutations causing A-T severe loss of function due to the severe truncation of proteins with many variants.
 
 
 
 
 
 
 
References
Adamovich AI. 2019. Functional analysis of BARD1 and BRCA1 variants of uncertain significance in homology-directed repair. (Doctoral dissertation, The Ohio State University).
Federici G, Soddu S. 2020. Variants of uncertain significance in the era of high-throughput genome sequencing: a lesson from breast and ovary cancers. Journal of Experimental & Clinical Cancer Research, 39(1), 1-12.
Findlay GM, Daza RM, Martin B, Zhang MD, Leith AP, Gasperini M, Janizek JD, Huang X
Starita LM, Shendure J. 2018. Accurate classification of BRCA1 variants with saturation genome editing. Nature. 562(7726): 217–222.
HHS Public Access: Accurate classification of BRCA1 variants with saturation genome editing. [accessed 2020 Oct. 22] file:///C:/Users/PC/AppData/Local/Temp/Temp3_archive%20(12).zip/2018_AccurateClassificationofBRCA1VariantswithSaturation.pdf.
Morash M, Mitchell H, Beltran H, Elemento O, Pathak J. 2018. The role of next-generation sequencing in precision medicine: A review of outcomes in oncology. J Pers Med. 8(3): E30.