CRISPR-Cas9 Gene Editing – Types, Components, Mechanism, Applications

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CRISPR-Cas9 was adapted from a naturally occurring genome editing system that bacteria use as an immune defense. When infected with viruses, bacteria capture small pieces of the viruses' DNA and insert them into their own DNA in a particular pattern to create segments known as CRISPR arrays. The CRISPR arrays allow the bacteria to "remember" the viruses (or closely related ones). If the viruses attack again, the bacteria produce RNA segments from the CRISPR arrays that recognize and attach to specific regions of the viruses' DNA. The bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which disables the virus.

Researchers adapted this immune defense system to edit DNA. They create a small piece of RNA with a short "guide" sequence that attaches (binds) to a specific target sequence in a cell's DNA, much like the RNA segments bacteria produce from the CRISPR array. This guide RNA also attaches to the Cas9 enzyme. When introduced into cells, the guide RNA recognizes the intended DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location, mirroring the process in bacteria. Although Cas9 is the enzyme that is used most often, other enzymes (for example Cpf1) can also be used. Once the DNA is cut, researchers use the cell's own DNA repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence.

What is CRISPR-Cas9?

• CRISPR-Cas9 is a genome-editing technique that is generating excitement within the scientific community. It is faster, less expensive, and more precise than earlier DNA editing approaches, and it has a wide range of possible uses.

• CRISPR-Cas9 is an innovative technology that allows geneticists and medical researchers to edit portions of the genome by removing, inserting, or modifying DNA sequence segments.

• In common usage, “CRISPR” (pronounced “crisper”) is an abbreviation for “CRISPR-Cas9.” CRISPRs are specific sequences of DNA, and the protein Cas9 — where Cas stands for “CRISPR-associated” — is an enzyme capable of severing DNA strands.

• Adapted from the natural defence mechanisms of bacteria and archaea, a category of relatively primitive single-celled microorganisms, CRISPR technology is a form of gene editing. Viruses are repelled by these species using CRISPR-derived RNA, a molecular cousin to DNA, and different Cas proteins.

• In order to thwart attacks, organisms sever the DNA of viruses and then store fragments of that DNA in their own genome to be deployed as a weapon if the viruses attack again.

• When the components of CRISPR are transferred into other, more complex organisms, these components can then manipulate genes, a process known as “gene editing.” No one knew what this process looked like until 2017, when a team of researchers led by Mikihiro Shibata of Kanazawa University in Japan and Hiroshi Nishimasu of the University of Tokyo revealed for the first time what a CRISPR looks like in action.

• It is currently the easiest, most adaptable, and most accurate approach of genetic manipulation, and is consequently generating a buzz in the scientific community.

• Comparable to an immune system, certain bacteria have an innate gene-editing mechanism similar to CRISPR-Cas9 that they use to respond to invading pathogens such as viruses.

• Using CRISPR, the bacteria remove portions of the virus’ DNA while retaining a portion of it to enable them recognise and defend against the virus in the future.

• Scientists modified this technique so that it could be employed in various animal cells, including those of humans and mice.

• What have scientists discovered about genetics throughout the years? and gene function by investigating the consequences of DNA mutations.

• If it is possible to alter a gene in a cell line or an entire organism, it is possible to examine the effect of this alteration in order to determine the function of the gene.

• Long ago, geneticists caused mutations using chemicals or radiation. However, there was no method to control where mutations occur in the genome.

• Scientists have been employing “gene targeting” for a number of years to induce alterations in specific locations of the genome by removing or inserting complete genes or single nucleotides.

• Traditional gene targeting has been very useful for researching genes and genetics, but creating a mutation is time-consuming and costly.

• Several ‘gene editing’ technologies, including CRISPR-Cas systems, transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases, have recently been created to improve gene targeting techniques (ZFNs).

• The CRISPR-Cas9 system is currently the most efficient, cost-effective, and trustworthy method for ‘editing’ genes.

What is gene/genome editing?

Manipulating the genetic makeup of an organism is referred to as gene-editing.

• Genome editing, also known as genome engineering or gene editing, is a form of genetic engineering involving the insertion, deletion, modification, or replacement of DNA in the genome of a living creature.

• Genome editing, in contrast to early genetic engineering approaches that randomly inserted genetic material into a host genome, directs the insertions to specified sites.

• Some prefer the term genome editing, but both names are acceptable; we can use either one. The primary purpose of DNA editing is to eliminate aberrant or flawed DNA sequences.

• In the late 1970s, Herb Boyer and Stanley Cohen found antibiotic-resistant genetically altered bacteria, which brought gene editing to light. Before 2012, the concept of gene editing was a myth.

• The discovery of the CRISPR gene editing technique in 2012 altered the era of genetics. A team of researchers from the University of California discovered the CRISPR-CAS9 system, a so-called bacterial immune system that can modify genes at the targeted spot.

• In 1982, however, human insulin-producing bacteria were identified and the concept evolved. After FDA approval, this synthetic insulin proved so popular that it became commercially available.

• Following the successful adoption of synthetic insulin, a road map was developed for the genetically modified Flvr shower tomato, bringer, and certain cotton species. This is a brief history and landmark of gene-editing technology.

• It is currently simple to edit the genes of bacteria, yeast, and mice, but not the human genome. Human embryo testing and research is not impossible, however there are certain ethical concerns involved with it. In the final section of this article, we’ll explore ethical concerns. The gene editing procedure consists of the following steps: Identification of a target spot inside an organism’s genome. Creating a healthy replica of the intended website. Conception of the directed RNA. A chemical process that cuts the flawed DNA. The addition of new DNA. Restoring the action site.

• The targeted region in the genome or gene is defective or mutated and produces defective or mutant protein. With the aid of the enzyme, the “Guided molecules” (which are nucleic acids) bind to the exact target spot within the genome.

• After binding of a guided molecule to the target site, the enzyme is a nuclease, a bacterial nuclease also known as a molecular scissor or an engineered nuclease that cuts DNA.

• Once the guided molecule binds to the region of action, the nuclease eliminates the DNA fragment from the genome. A nick is produced at this location, which is then repaired by the cell’s natural DNA repair system.

• During the replication process, the cell’s natural DNA repair system identifies any mutations or breaks in the genome and fills them with complimentary nucleotides. We can also introduce the DNA fragment of interest at that exact location.

• Here, the guided molecule is a gRNA or sgRNA RNA (single-stranded RNA). We have authored a paper on the structure, function, and design of gRNA.

• Using nuclease-controlled gene editing, we can remove or insert the desired DNA sequence. This is a straightforward explanation of gene editing. The illustration of gene editing is depicted in the figure below.

What is the need for gene-editing?

• Almost all genetic illnesses are fatal and incurable. Following the discovery of DNA in 1953, the inheritance of disease becomes clearer. The chromosome carries the inherited DNA from generation to generation.

• Thus, genetic material is transmitted from parents to kids, and so are mutations! Any modifications to a genome can be transmitted to progeny. And a genetic mutation occurs. This mutated region of the genome can be removed using gene editing.

Importance of gene editing

• It can be used to change the genome of any creature, but it is particularly useful for bacteria, yeast, mice, and other model species.

• It is used to investigate the gene expression of living organisms.

• The approach can eliminate mutated genes from an organism’s genome and also be used to generate new variants.

• It is possible to alter the properties of an organism.

• Additionally, it is possible to develop genetically engineered organisms with economic value.

Types of gene-editing

The genetically engineered nuclease enzyme molecular scissors serves a crucial role in the gene-editing process. Therefore, depending on the type of nuclease, numerous genetic engineering techniques have been in use for decades. There are three major techniques listed here:

• ZFNs (Zinc finger nuclease)

• TALEN (Transcriptional activator-like Effector based Nuclease)

• CRISPR-CAS9 (clustered regularly interspaced short palindromic repeats)

What is TALENs?

• Transcription activator-like effector nucleases (TALENs) are DNA-binding proteins with an array of 33 or 34 repeating amino acids.

• TALENs are synthetic restriction enzymes created by combining the DNA-cutting domain of a nuclease with TALE domains, which may be programmed to identify a specific DNA sequence.

• These fusion proteins serve as readily targetable “DNA scissors” for gene editing applications, enabling targeted genome modifications in living cells, including sequence insertion, deletion, repair, and replacement.

• The DNA binding domains, which may be engineered to bind any desired DNA sequence, are derived from TAL effectors, which are DNA-binding proteins excreted by the plant-pathogenic Xanthomanos spp.

• TAL effectors are composed of repetitive domains, each containing a highly conserved sequence of 34 amino acids, and they detect a single DNA nucleotide within the target region.

• The nuclease is capable of creating double strand breaks at the target location, which can be repaired by error-prone non-homologous end-joining (NHEJ), resulting in gene disruptions caused by the introduction of tiny insertions or deletions. Except for the so-called repeat variable di-residues (RVDs) at amino acid positions 12 and 13, each repeat is conserved.

• RVDs are responsible for determining the DNA sequence to which the TALE will attach. This clear one-to-one correlation between TALE repeats and the appropriate DNA sequence simplifies the process of constructing repeat arrays to identify novel DNA sequences.

• These TALEs can be joined with the catalytic domain of the DNA nuclease FokI to produce a transcription activator-like effector nuclease (TALEN). Combining specificity with activity, the resultant TALEN constructions provide tailored sequence-specific nucleases that bind and cleave DNA sequences only at predetermined places.

• The TALEN target recognition system utilises a code that is simple to predict. In part because to the length of their 30+ base pair binding site, TAL nucleases are unique to their target. Within a range of six base pairs, TALEN may modify any single nucleotide in the whole genome.

• Similar to engineered zinc finger nucleases, TALEN constructions have three advantages in targeted mutagenesis: (1) improved DNA binding specificity, (2) lesser off-target effects, and (3) faster assembly of DNA-binding domains.

 

What is ZFNs?

• Zinc-finger nucleases (ZFNs) are synthetic restriction enzymes made by combining a zinc finger DNA-binding domain with a DNA-cleavage domain.

• The ability to build zinc finger domains to target specific DNA sequences enables zinc-finger nucleases to target unique sequences within complicated genomes.

• These reagents can be used to precisely modify the genomes of higher species by utilising endogenous DNA repair machinery.

• In addition to CRISPR/Cas9 and TALEN, ZFN is a significant technology for genome editing.

• Zinc finger nucleases are beneficial for manipulating the genomes of numerous plants and animals, such as arabidopsis, tobacco, soybean, corn, Drosophila melanogaster, and Candida albicans. elegans, Platynereis dumerilii, sea urchin, silkworm, zebrafish, frogs, mice, rats, rabbits, pigs, and cattle, as well as many mammalian cell types.

• Zinc finger nucleases have also been employed in a mouse model of haemophilia, and a clinical experiment indicated that CD4+ human T-cells with the CCR5 gene damaged by zinc finger nucleases are safe as a potential HIV/AIDS treatment.

• ZFNs are also employed to produce isogenic human illness models, a new generation of genetic disease models.

Components Of Crispr

1. CRISPRs

• CRISPR is an acronym for “clusters of regularly interspaced short palindromic repeats” and it refers to a section of DNA composed of short, repeating sequences with so-called “spacers” placed between each repeat.

• Repeats in the genetic code refer to the ordering of rungs within the spiral ladder of a DNA molecule. Each rung comprises two chemical bases linked together: adenine (A) is linked to thymine (T), and guanine (G) is linked to cytosine (C) (C).

• According to the Max Planck Institute, in a CRISPR area, these bases are repeated in the same order multiple times, forming what are known as “palindromic” sequences.

• In a palindromic sequence, the bases on one side of the DNA ladder match those on the opposite side when they are read in opposite directions.

• A highly simple palindromic sequence might seem like follows: GATC, CTAG

• Short palindromic repeats are found throughout CRISPR sections of DNA, with each repeat flanked by “spacers.” Bacteria steal these spacers from viruses that have attacked them, so incorporating a portion of viral DNA into their own genome.

• These spacers function as a memory bank, allowing the bacteria to recognise the viruses should they ever strike again. You may also compare spacers to “Wanted” posters, as they provide a snapshot of the criminals, allowing them to be readily identified and brought to punishment.

• This procedure was originally experimentally proved by Rodolphe Barrangou and a team of researchers at Danisco, a manufacturer of food additives.

• The researchers utilised Streptococcus thermophilus bacteria, which are often found in yoghurt and other dairy cultures, as their model in a 2007 paper published in the journal Science, according to the Joint Genome Institute, part of the U.S. Ministry of Energy.

• After a viral infection, the bacteria were observed to incorporate additional spacers into their CRISPR regions. In addition, the DNA sequence of these spacers was identical to that of portions of the virus genome.

• Additionally, the team removed the spacers and inserted fresh viral DNA sequences in their place. In this manner, the researchers were able to modify the bacteria’s resistance to a particular virus, proving the significance of CRISPRs in regulating bacterial immunity.

2. CRISPR RNA (crRNA)

• CRISPR sections of DNA serve as a sort of memory bank for viral information; however, for this information to be useful elsewhere in the cell, it must be replicated, or “transcribed,” into RNA.

• Unlike DNA sequences, which remain lodged within the DNA molecule, this CRISPR RNA (crRNA) can roam throughout the cell and collaborate with proteins, namely the molecular scissors that snip viruses to pieces.

• RNA varies from DNA in that it consists of a single strand, as opposed to two, giving it the appearance of half a ladder.

• To generate an RNA molecule, one portion of CRISPR serves as a template, and proteins known as polymerases construct an RNA molecule that is “complementary” to the template, meaning that the bases of the two strands fit together like puzzle pieces. A G in the DNA molecule, for instance, would be translated as a C in the RNA.

• According to a 2014 assessment published in the journal Science by Jennifer Doudna and Emmanuelle Charpentier, each snippet of CRISPR RNA comprises a copy of a repeat and a spacer from a CRISPR region of DNA.

• The crRNA interacts with the Cas9 protein and another type of RNA, known as “trans-activating crRNA” or tracrRNA, to aid bacteria in their fight against viruses.

3. Cas9

• Cas9 is an enzyme capable of severing foreign DNA. The protein attaches to crRNA and tracrRNA, which together direct Cas9 to the spot on the viral DNA strand where it will make its cut.

• Cas9 will cut through target DNA that is complementary to a 20-nucleotide length of the crRNA, where a “nucleotide” is a single-base DNA building block.

• Using two distinct sections or “domains” on its structure, Cas9 cuts both strands of the DNA double helix, creating a “double-stranded break,” according to a 2014 research published in Science.

• Cas9 is equipped with a safety mechanism that prevents it from randomly slicing a genome. PAMs, or protospacer adjacent motifs, are short DNA sequences that function as tags that are located adjacent to the target DNA sequence.

• If there is no PAM adjacent to the target DNA sequence, Cas9 will not cut. This is one possible explanation for why Cas9 never attacks the CRISPR region in bacteria, according to a 2014 Nature Biotechnology review.

CRISPR-CAS9 gene editing system

• Some bacteria and archaea contain CRISPR- Clustered Regularly Interspaced Short Palindromic Repeats. It is a component of the natural defensive mechanism of bacteria. The RNA generated from CRISPR protects bacteria against invading viruses and phages.

• The nuclease, CAS9 protein cuts and destroys the DNA strand of invading infections.

• In 1980, the gene sequences for CRISPR were identified in E. coli, although their function was unknown at the time. Later, in 2007, Barrangou et al. reported their function in the bacteria’s adaptive immune system.

CRISPR-CAS9 in Bacteria

• Throughout the viral infection, the virus injects its DNA through the bacterial cell wall.

• The DNA of the phage is integrated into the host genome and then transcribed and translated.

• The viral protein is generated from the bacterial DNA.

• To escape phage infection, bacteria have created a distinctive defence mechanism known as a CRISPR-CAS9-based defence mechanism.

• During the viral invasion, the bacteria added reference DNA sequences into the palindromic repeats that create CRISPR. Combining bacterial palindromic repeat sequences and viral spacer DNA creates CRISPR.

• The spacer DNA is transcribed into the RNA called crRNA, which directs viral genome binding at a precise site. The DNA-RNA heteroduplex is formed at the invasion site.

• The bacteria also produced a number of short tracrRNA (trans-activating crRNA), which are partially complementary to the crRNA.

• The tracrRNA facilitates the maturation of numerous pre-crRNA into mature crRNA.

• gRNA or guided RNA is the junction of crRNA and tracrRNA that directs the nuclease to cut the target sequence.

• This gRNA is recognised by CAS9, which binds to the site of the heteroduplex. Two unrelated domains of a nuclease (CAS9 nuclease) designated a RuvC-like domain and HNH domains lyses the phage dsDNA after it is suitably positioned.

• Throughout this procedure, the CRISPR sequences serve as a target for nuclease to cut the DNA. The DNA of the virus or phase cannot reproduce and destroy.

• The RuvC domain severing the non-homologous domain, whereas the HNH domain severing the homologous domain (homologous to gRNA).

• CRISPR and CAS9 protein work in concert to eliminate the invading pathogen or virus. This theory raises the question, “Does it cut its own DNA? The response is “Yes”.

• The viral DNA sequence is also found in CRISPR. Therefore, it is possible that CRISPR-CAS9 detects these spacer sequences as foreign DNA and edits them accordingly. Actually, it does not occur because of a pattern known as a PAM!

• The photo spacer-associated motif (PAM) aids in gRNA recognition by CAS9. The nuclease is situated close to the PAM domain. It binds to the 3′-OH terminus of gRNA.

• This will prompt CAS9 to begin cleaving the DNA. CAS9 detects the PAM-gRNA complex on double-stranded DNA and catalyses the process. Lack of PAM in the DNA of bacteria protects them. It is a type of safety mechanism designed by bacteria themselves.

• PAM safeguards bacterial DNA against its own nuclease activity.

• CRISPR are the DNA sequences that are composed of spacers and repeats. The spacers are present throughout the CRISPR between the short palindromic repeats. Different spacer sequences are extracted from various viruses to aid bacteria during future pathogen attacks.

• These sequences contain information that bacteria employ to eliminate pathogens. But how can we put it to use?

• We are able to construct synthesised gRNA that directs CAS9 to the desired target site, allowing us to conduct a variety of genome-editing experiments.

CRISPR-CAS9 in genetic engineering

• Guided RNA and the dimeric CAS9 protein are the only prerequisites for gene editing.

• Once the nuclease has cleaved the DNA at the designated place, it generates the double-stranded cut. Now, the cell’s natural DNA repair system utilises either non-homologous end-joining or Homologous direct repair to close the gap.

• The entire system functions as a result of double-stranded break repair.

Non-homologous end-joining

• In this form of DNA repair process, the gap is directly closed by uniting two non-homologous strands that are opposite one another. Several essential pieces of genetic information may be lost in NHEJ because the whole DNA fragment is missing.

• This DNA fragment will never be present in the subsequent cell division, causing a mutation in the genome. The condition becomes fatal for the organism if some of its essential genes are eliminated.

Homologous direct repair

• In homologous repair, the gaps are not directly closed. Homologous direct repair follows the recombination mechanism. Here, the nucleotides are inserted at the cut spot depending on the data from the previous replication.

• The cell employs single-stranded DNA as a template for the insertion of additional nucleotides throughout this process. Now is the moment to introduce our desired single-stranded DNA, which is generated spontaneously by the cell throughout successive cell divisions.

• By using non-homologous end joining, we can eliminate defective DNA while homologous direct repair can insert DNA.

• CRISPR-CAS9 is utilised in molecular genetics and genetic engineering as a gene-editing technology. On the basis of the activity of CAS9, three distinct gene-editing systems are hypothesised. The following are the three types of systems: CAS9 with cleavage activity only, Full CAS9 nuclease activity, CAS without the activity of cleavage.

• In the first approach, the CAS9 encoding gene is used to generate the cas9D10A mutant. This mutant is incapable of severing both strands of DNA. Instead, cas9D10A recognises the gRNA and only cleaves one strand of the target DNA.

• What are the advantages of this mutant? In fact, it improves the accuracy of gene editing. It permits only high-fidelity homologous direct repair of the single-stranded gap, hence decreasing the likelihood of deletion-insertion mutations in the gnome.

• In brief, the cas9D10A variation prohibits non-homologous end-joining.

• The second mechanism is CRISPR-CAS9 functioning normally. None of the sequences for CAS9 proteins are altered. We have already described the CRISPR-CAS9 technology and how standard CAS9 operates.

• The third system relies on the dCas9 gene. dCas9 is often referred to as nuclease-deficient CAS9 or dead Cas. Some mutations induced artificially into the RuvC and HNH domains inhibit the function of nuclease.

• The nuclease-deficient CAS9 cannot cut the target sequence; it can only bind to it. With the aid of several effector domains, this permits the silencing or activation of a certain gene.

 

Application of CRISPR-CAS9

CRISPR gene editing

• In the food and agriculture industries, CRISPR has been used to engineer probiotic cultures and immunise industrial cultures (such as yoghurt) against illnesses. It is also used to increase crop output, drought resistance, and nutritional value.

• By the end of 2014, approximately one thousand research papers mentioning CRISPR had been published.

• The approach had been used to functionally inactivate genes in human cell lines and cells, to research Candida albicans, to genetically edit agricultural strains, and to modify yeasts used to produce biofuels.

• According to Hsu and his colleagues, the capacity to adjust genetic sequences enables reverse engineering, which has a favourable impact on biofuel production. CRISPR can also be used to modify mosquitos so that they cannot spread diseases such as malaria.

• Recently, a large variety of plant species have been successfully modified using Cas12a-based CRISPR techniques.

• A genetic disease patient was experimentally treated with CRISPR in July 2019. A 34-year-old lady with sickle cell illness was the patient.

• Using LASER ART, a novel antiretroviral medicine, and CRISPR, sixty to eighty percent of the integrated viral DNA was eliminated from animals in February 2020, and some mice were fully free of the virus.

• In an effort to treat Leber congenital amaurosis, a patient’s eye was injected with a CRISPR-modified virus in March 2020.

• CRISPR gene editing could potentially be utilised in the future to create new species or resurrect extinct species from closely related populations.

• Reevaluations of gene-disease correlations using CRISPR have led to the discovery of potentially significant abnormalities.

CRISPR as diagnostic tool

• Due to their capacity to specifically target nucleic acid sequences against a high background of non-target sequences, CRISPR-associated nucleases have proven effective as a tool for molecular testing.

• In 2016, the Cas9 nuclease was utilised to remove undesirable nucleotide sequences from next-generation sequencing libraries with only 250 picograms of RNA input.

• In 2017, CRISPR-associated nucleases were also employed for direct diagnostic testing of nucleic acids, with sensitivity down to a single molecule. By Martins et al. (2019), CRISPR diversity is utilised as an analytic target to determine phylogeny and diversity in bacteria, such as xanthomonads.

• As established by Shen et al., 2020, early identification of plant infections by molecular typing of the pathogen’s CRISPRs can be utilised in agriculture.

• By integrating CRISPR-based diagnostics with additional enzymatic activities, it is possible to identify substances other than nucleic acids. SHERLOCK-based Profiling of IN vitro Transcription is one example of a linked technology (SPRINT). SPRINT can be used to identify a number of compounds, such as metabolites in patient samples or environmental toxins, with high throughput or with portable point-of-care devices.

• CRISPR/Cas platforms are also being investigated for the detection and elimination of SARS-CoV-2, the virus responsible for COVID-19.

• For SARS-CoV-2, the AIOD-CRISPR and SHERLOCK assays have been identified as thorough diagnostic options.

• The SHERLOCK test is based on a press reporter RNA that is fluorescently labelled and capable of identifying 10 copies per microliter. AIOD-CRISPR facilitates robust and sensitive optical detection of viral nucleic acid.

 

Limitations of CRISPR-Cas9 system

CRISPR/Cas is a highly effective technique, yet it has significant limitations. It is:

• It is difficult to transfer large quantities of CRISPR/Cas material to mature cells, which is a difficulty for many therapeutic applications. Viral vectors are the most prevalent method of transmission.

• not 100% effective, therefore even cells that take in CRISPR/Cas may not change their genomes.

• Not one hundred percent accurate, and “off-target” modifications, although uncommon, can have serious repercussions, especially in therapeutic applications.

Potential Risks And Ethical Concerns Of Using Crispr

• Numerous possible applications of CRISPR technology raise problems regarding the ethics and repercussions of genome modification.

• The 2018 announcement by He Jiankui, a former biophysicist at the Southern University of Science and Technology in Shenzhen, that his team had modified DNA in human embryos and therefore generated the world’s first gene-edited newborns sparked numerous ethical arguments.

• Live Science earlier reported that he was sentenced to three years in prison and fined 3 million yuan for practising medicine without a licence, violating Chinese restrictions on human-assisted reproductive technology, and forging ethical review documents. However, even after his punishment, He’s experiments sparked questions about how the future use of CRISPR should be governed, given the relative youth of the technology.

• Obviously, illegal research on human embryos is an extreme misuse of CRISPR, but scientists assert that even ostensibly legitimate applications of the technology contain risks.

• Germline editing is the process of applying genetic adjustments to human embryos and reproductive cells, such as sperm and eggs. As modifications to these cells can be passed on to future generations, the use of CRISPR technology to modify the germline has raised a variety of ethical concerns.

• Safety issues are posed by variable efficacy, off-target consequences, and inaccurate editing. In addition, there is still much that the scientific community does not understand. In a 2015 article published in Science, David Baltimore and a group of scientists, ethicists, and legal experts note that germline editing may have unintended consequences for future generations “due to our limited understanding of human genetics, gene-environment interactions, and the pathways of disease” (including the interplay between diseases or conditions in the same patient).

• In a 2014 study published in Science, Oye and colleagues discuss the potential ecological effects of gene drives. Through crossbreeding, an introduced trait could extend beyond the intended population to other creatures. Additionally, gene drives could limit the genetic variety of the target population, which could hinder its ability to thrive.

• Other ethical issues are complicated in nature. Should we make significant changes that could affect future generations without their consent? What if germline editing evolves from a therapeutic tool to a tool for enhancing various human characteristics?

• To address these concerns, the National Academies of Sciences, Engineering, and Medicine have compiled a detailed report with recommendations and guidelines for genome editing.

• The National Academies advocate caution in the pursuit of germline editing, but they underscore that “caution does not mean prohibition.” They recommend that germline editing be performed only on genes that contribute to significant disorders and only when there are no other viable therapy options. They emphasise, among other things, the need to collect information on the risks and benefits to health and to maintain constant oversight during clinical trials. In addition, they urge that, following the conclusion of a trial, trial organisers follow up with the participants’ relatives for numerous generations to see which alterations in the genome remain over time.

Who Discovered Crispr?

• According to Quanta Magazine, scientists initially identified CRISPRs in bacteria in 1987, but they did not comprehend the biological importance of the DNA sequences and did not yet label them “CRISPRs” (opens in new tab).

• Yoshizumi Ishino and his colleagues at Osaka University in Japan discovered the distinctive nucleotide repeats and spacers in the intestinal bacteria Escherichia coli. As genetic analysis technology advanced in the 1990s, other researchers discovered CRISPRs in a variety of other microbes.

• According to a 2016 report(opens in new tab) in the journal Cell, Francisco Mojica, a researcher at the University of Alicante in Spain, was the first to characterise the unique characteristics of CRISPRs and discover the sequences in 20 different bacteria. Initially, he referred to the sequences as “short regularly spaced repeats” (SRSRs), but later he proposed that they be referred to as CRISPRs instead. Mojica has been in touch with Ruud Jansen of Utrecht University, who coined the name CRISPR in a 2002 article published in the journal Molecular Microbiology.

• In the years that followed, scientists also found Cas genes and the function of Cas enzymes, as well as the origin of the spacers in CRISPRs, as reported by Quanta.

• Jennifer Doudna, a professor of biochemistry, biophysics, and structural biology at the University of California, Berkeley, and Emmanuelle Charpentier, the director of the Max Planck Unit for the Science of Pathogens, shared the Nobel Prize in chemistry in 2020. Previously reported by Live Science, the two scientists are credited with turning the bacterial CRISPR/Cas system into a useful gene-editing tool.

• Charpentier discovered tracrRNA while studying the bacteria Streptococcus pyogenes, which is responsible for a variety of illnesses ranging from tonsillitis to sepsis. Charpentier began collaborating with Doudna to construct the CRISPR/Cas system when tracrRNA was identified as a previously unknown component. In 2012, the group announced in the journal Science that they had effectively transformed the molecular scissors into a gene-editing tool in their seminal work.

• Science Magazine noted that others believed Feng Zhang, a scientist at the Broad Institute, may also win the Nobel Prize for his independent contributions to the CRISPR system. Zhang revealed that the CRISPR system functions in mammalian cells; thus, the Broad Institute was awarded the first patent for the use of CRISPR gene-editing technology in eukaryotes, or complex cells with nuclei that contain DNA.

 

How Crispr Works As A Genome-Editing Tool described in Summary?

• Genomes encode a set of messages and instructions inside their DNA sequences. Genome editing entails modifying these DNA sequences, hence altering the encoded messages. This can be accomplished by placing a cut or break in the DNA and deceiving the cell’s normal DNA repair machinery into making the desired modifications. CRISPR-Cas9 provides a mechanism to do so.

• In 2012, two major scientific articles were published in the journals Science and PNAS that described how the bacterial CRISPR-Cas9 could be used to cut up any DNA, not simply that of viruses. Thus, the natural CRISPR system might be converted into a straightforward, programmable genome-editing instrument.

• Scientists may simply alter the sequence of the crRNA, which binds to a complementary sequence in the target DNA, the research concluded.In the 2012 Science publication, Martin Jinek and his colleagues further simplified the technique by merging crRNA and tracrRNA to create a single “guide RNA.” Thus, genome editing requires only two components: a guide RNA and the Cas9 protein.

• George Church, a professor of genetics at Harvard Medical School, told Live Science, “Operationally, you create a stretch of 20 base pairs that match a gene that you wish to alter.” From there, one may determine the complementary crRNA sequence. Church underlined the need of ensuring that the nucleotide sequence is unique to the target gene and not present anywhere else in the genome.

• Church added, “then the RNA plus the protein [Cas9] will cut, like a pair of scissors, the DNA at that place, and ideally nowhere else.” Once the DNA is cut, the cell’s inherent repair mechanisms kick in and begin to reassemble the DNA; at this stage, genome modifications are possible. There are two possible outcomes:

• According to the Stanford University Huntington’s Outreach Project, one form of mending involves glueing the two cuts back together. This technique, termed as “non-homologous end joining,” has a tendency to introduce errors in which nucleotides are inadvertently added or deleted, resulting in mutations that may disrupt a gene.

• In the second procedure, the break is repaired by inserting a nucleotide sequence into the gap. To accomplish this, the cell employs a short DNA strand as a template. Scientists can provide the DNA template of their choosing, allowing them to insert any gene or fix a mutation.

 

References

Ran, F., Hsu, P., Wright, J. et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8, 2281–2308 (2013). https://doi.org/10.1038/nprot.2013.143

https://www.yourgenome.org/facts/what-is-crispr-cas9/

https://crisprtx.com/gene-editing/crispr-cas9

https://www.jax.org/personalized-medicine/precision-medicine-and-you/what-is-crispr

https://en.wikipedia.org/wiki/Genome_editing

https://geneticeducation.co.in/what-is-gene-editing-and-crispr-cas9/

https://www.livescience.com/58790-crispr-explained.html