Monday, March 29, 2021

How to study point mutation with gene editing in cell lines | Ubigene

 



What is point mutation? A point mutation is specifically when only one nucleotide base is changed in some way.

Types of point mutations: Point mutation can be classified based on their location and function.

Based on location:


1.Coding region

a. A point mutation will commonly result in the same amino acid being incorporated into the resulting polypeptide despite the sequence change. This change would have no effect on the protein’s structure and is thus called a silent mutation. 

b. A missense mutation results in a different amino acid being incorporated into the resulting polypeptide. The effect of a missense mutation depends on how chemically different the new amino acid is from the wild-type amino acid. 

c. A nonsense mutation that converts a codon encoding an amino acid (a sense codon) into a stop codon (a nonsense codon). Nonsense mutations result in the synthesis of proteins that are shorter than the wild type and typically not functional.


2.Non-coding region: There are in fact many ways how mutations in "intra-genic" non-coding regions can (and do) affect the expression and function of the gene.

a. The introns may contain sequences that bind additional transcriptional enhancers or silencers (but not necessary for the gene that contains these elements), so mutations in these regions can also influence the transcription.

b. Introns also contain sequences of regulatory RNAs (miRNAs, lincRNAs) that may affect the translation and stability of the mRNA of the including (and/or other) gene(s). When the mutations change the processing or sequence of these RNAs this can surely change the amount produced of gene product.

c. A further level of regulation applies during splicing. This may be altered when the splicing signals in the introns are mutated, where the splice factors bind. Mutations in these regions may lead to differentially spliced or truncated products, that may not be functional or even have different functions.

d. At the level of translation, the UTRs are involved in regulating the activity of protein production, so mutations in these regions may affect the amount of protein produced.


Based on functional effect:

1. Loss of function: In a wild-type diploid cell, there are two wild-type alleles of a gene, both making normal gene product. But in heterozygotes, the single wild-type allele may be able to provide enough normal gene product to produce a wild-type phenotype. In such cases, loss-of-function mutations are recessive. In some cases, the cell can “upregulate” the level of activity of the single wild-type allele so that in the heterozygote the total amount of wild-type gene product is more than half that found in the homozygous wild type. However, some loss-of-function mutations are dominant. In such cases, the single wild-type allele in the heterozygote cannot provide the amount of gene product needed for the cells and the organism to be wild type. Thus, loss of function means the gene product having less or no function (being partially or wholly inactivated).

2. 2.Gain of function: A type of mutation in which the altered gene product possesses a new molecular function or a new pattern of gene expression. Gain-of-function mutations are almost always dominant or semidominant. But in case of heterozygote, the new function will be expressed, and therefore the gain-of-function mutation most likely will act like a dominant allele and produce new phenotype.


How to study point mutation with gene editing in cell lines:


Gene editing technology, CRISPR/cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models for human diseases, for example, in-vitro cell lines and in human pluripotent stem cells (hiPSC). With modern sequencing, CRISPR technologies can introduce precise point mutations (homozygous/heterozygous) in various cell lines and able to generate a human disease model, which is useful for disease therapeutics and basic genomic studies. Over the years, the cellular model has proven to be useful in reproducing human disease.


CRISPR/cas9 can introduce targeted double-stranded break (DSBs) with high efficiency. These DSBs can be repaired by a homology-directed repair (HDR) system by using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knockin of specific mutations. CRISPR based knock-in genome-editing framework allows researchers to selective introduction of mono- and bi-allelic sequence changes as well as pathogenic disease modeling of heterozygous and homozygous point mutations. Homozygous point mutations require a guide RNA targeting close to the intended mutation, whereas heterozygous can be accomplished by distance-dependent suboptimal mutation incorporation or using mixed repair templates. CRISPR-mediated knock-in point mutations are extremely useful for gain-of-function (GOF) or loss of function (LOF) studies. CRISPR-mediated knock-in point mutation has enormous potential to improve our prognostic capacity of patients affected by this disorder and may ultimately open new avenues for disease interrogation and targeting.


Due to available genetic engineering tools, most researcher willing to develop cellular model for human diseases to understand disease developed mechanism and potential therapeutics.


Model that’s required heterozygous genotype:


In diploid organisms, a mutation that occurs on only one allele for a gene is called a heterozygous mutation. Heterozygous just means that a person has two different versions of the gene (one inherited from one parent, and the other from the other parent). In diseases caused by what are called dominant genes, a person needs only one disease-causing copy of a gene to have problems. A person with only one affected gene (inherited from either parent) will still almost certainly get disease due to the gene dominance nature. Thus, if a dominant gene causes a disease, a heterozygote may manifest the disease. If a recessive gene causes a disease, a heterozygote may not develop the disease or may have lesser effects of it. An example of heterozygous point mutation related disease centronuclear myopathy.


Centronuclear myopathy is a condition characterized by muscle weakness (myopathy) and wasting in the skeletal muscles. People with centronuclear myopathy begin experiencing muscle weakness at any time from birth to early adulthood. The muscle weakness slowly worsens over time and can lead to delayed development of motor skills, such as crawling or walking; muscle pain during exercise; and difficulty walking. Some affected individuals may need wheelchair assistance as the muscles atrophy and weakness becomes more severe. In rare instances, muscle weakness improves over time. Centronuclear myopathy is most often caused by mutations in the DNM2, BIN1, or TTN gene. When centronuclear myopathy is caused by mutations in the DNM2 gene, it is inherited in an autosomal dominant pattern, which means one copy of the altered DNM2 gene in each cell is sufficient to cause the disorder. DNM2-related CNM is mainly caused by heterozygous single point mutations. The CGG codon in humans’ codes for a conserved arginine residue at amino acid position 465 but in patients Dnm2 R465 CGG codons are changed into TGG, encoding a tryptophan.


Alzheimer's disease (AD) is a devastating neurodegenerative disease accounting for 50–75% of all forms of dementia. Approximately 44 million people worldwide were estimated to be diagnosed with AD or related dementia in 2015. Approximately 4.6 million new cases of dementia are reported annually, and the number of AD patients is expected to nearly double by 2030. Genetic factors may explain many of the variations affecting AD risk, particularly familial AD and early-onset AD (EOAD), in which most genetic variants are related to amyloid-β (Aβ) processing. EOAD is a subtype of AD in which disease onset occurs before the age of 65 years, but several patients develop AD in their 30 s or 40s. Three genes have been identified as causative factors for EOAD: amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2). AD is one of the heterozygous point mutation-related diseases.


Models that required Homozygous genotype:


Identical mutations that occur on both alleles of the same gene are called homozygous mutations. An example of homozygous point mutation disease is Tay-Sachs disease (TSD). TSD is a fatal autosomal recessive genetic disorder, most commonly occurring in children. TSD is caused by mutations in the HEXA (hexosaminidase-A) gene localized on chromosome 15. Without normal (hexosaminidase-A) gene localized on chromosome 15. Without normal HEXA, a fatty substance, or lipid, called GM2 ganglioside, accumulates abnormally in cells, especially in the nerve cells of the brain. This ongoing accumulation causes progressive damage to the cells and leads to a neurodegenerative disease. Nearly 130 mutations have been reported in the HEXA gene to cause TSD and its variants, including single base substitutions, small deletion, duplications, and insertions splicing alterations, complex gene rearrangement, and partial large duplications. In the Ashkenazi Jews, 94%–98% patients are caused by one of the three common mutations c.1277_1278insTATC, c.1421 + 1 G > C and c.805 G > A (p.G269S). The c.805 G > A (p.G269S) mutations also found in non-Ashkenazi Jewish populations, along with an intron 9 splice site mutation (c.1073 + 1 G > A). There is currently no treatment or cure.


Cystic Fibrosis (CF) is a recessive inherited disorder most common among people of European descent. In the United States, 1 in 3500 newborns are born with cystic fibrosis, and 1 in 30 Caucasian Americans is a carrier. There are many different mutations that can cause CF, but the most common one is a deletion of three nucleotides in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that results in the loss of the amino acid phenylalanine and causes an incorrectly folded protein. (Note that this deletion is not a frameshift mutation because three bases next to each other are deleted, and all the other amino acids in the chain remain the same.) CF is associated with thick, sticky mucus in the lungs and trouble breathing, salty sweat, infertility in certain individuals, and a shortened life expectancy (about 42-50 years in developed countries).

Ubigene Bioscience developed CRISPR/Cas9 technology-based CRISPR-U™ pipeline for gene-editing cell line generation. We provide versatile strategies including HDR mediated and non-HDR mediated for efficient point mutation, which will help you develop good cellular models for disease-related research.

No comments:

Post a Comment

[Research highlight] Enhancing p53 pathway can efficiently suppress colon cancer

  Colorectal cancer is the third most diagnosed cancer and leads to the second mortality among cancers worldwide. The first-line chemotherap...