CRISPR Gene Editing

CRISPR gene editing is a molecular biology technology that allows for the modification of living organisms' genomes. It is pronounced "crisper" (/krspr/). It is based on the CRISPR-Cas9 antiviral defence system used by bacteria. The genome of a cell can be sliced at a specific spot by introducing the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into the cell, enabling the addition of new genes or the deletion of existing ones in vivo.

The technique is fundamental in biotechnology and medicine because it enables precise, low-cost, and straightforward in vivo genome modification. It can be used to create novel drugs, foods, and genetically modified organisms in addition to controlling diseases and pests. Both somatic mutation-related illnesses like cancer and genetic abnormalities that run-in families may benefit from its use. However, its applicability to manipulating human germline genetic material is highly disputed. The technique was created by Jennifer Doudna and Emmanuelle Charpentier, who shared the 2020 Nobel Prize in Chemistry. The Kavli Prize for the same discovery was shared by a third research team led by Virginijus Iknys, although they did not get the Nobel Prize.

The Cas9 nuclease acts as a pair of genetic scissors, opening both strands of the DNA sequence that is being targeted to deliver the change using one of two strategies. The conventional process of targeted genome editing approaches involves knock-in mutations, which are assisted by homology directed repair (HDR). The introduction of targeted DNA damage and repair is made possible by this. By using foreign DNA to serve as the repair template, HDR uses comparable DNA sequences to propel the repair of the break. For the repair to start using this technique, periodic, isolated instances of DNA damage must occur at the target spot. The knock-out mutations induced by CRISPR-Cas9 are caused by the repair of the double-stranded break by non-homologous end joining (NHEJ) or POLQ/polymerase theta-mediated end-joining (TMEJ). End-joining pathways frequently result in random deletions or insertions at the repair site, which might affect or alter the functionality of genes. Therefore, tailored random gene disruption can be produced by researchers using CRISPR-Cas9 genome engineering. The accuracy of genome editing is therefore a major concern. The genome is altered irreversibly through genomic editing.

Since the 1980s, it has been possible to modify the genome in eukaryotic cells using a variety of techniques. However, these techniques have proven to be ineffective and difficult to use on a wide scale. Effective and incredibly selective editing is now a reality thanks to the discovery of CRISPR, and more especially the Cas9 nuclease protein. Streptococcus pyogenes derived Cas9 has enabled targeted genomic modification in eukaryotic cells by giving a reliable means to make a break at a specific location as suggested by the crRNA and tracrRNA guide strands. The convenience of using Cas9 and template RNA to silence or produce point mutations at loci has made it possible to quickly and effectively map the genomic models and biological processes connected to different genes in a range of eukaryotes. Cas9 nuclease variants that have undergone recent engineering to considerably minimise off-target activity have been created.

There are numerous potential uses for CRISPR-Cas9 genome editing technologies, including in agriculture and health. The AAAS selected genome editing with the CRISPR-Cas9-gRNA complex as the Breakthrough of the Year in 2015. The idea of employing CRISPR for germline editing has generated a lot of bioethical issues, especially when applied to human embryos.

Genome Engineering

Genome editing using CRISPR-Cas9 is possible with a Type II CRISPR system. When utilised for genome editing, this system contains a ribonucleoprotein (RNP) made up of Cas9, crRNA, and tracrRNA as well as an optional DNA repair template. Plasmids containing the RNP's component parts are frequently used by CRISPR-Cas9 to transfect the target cells, or the RNP is constructed before being supplied to the cells by nucleofection. The basic components of this plasmid are listed in both the table and the picture. As the sequence that Cas9 employs to recognise and directly bind to specific sequences inside the host cell's DNA, the crRNA is specifically created for each application. The crRNA must only bind to the sections that are intended for editing. Each application's repair template is specially created since it needs to partially complement the DNA sequences on either side of the cut as well as contain the necessary sequence for insertion into the host genome.

By combining several crRNAs with the tracrRNA, a single-guide RNA (sgRNA) can be produced. This sgRNA can be coupled with the gene encoding the Cas9 protein and produced into a plasmid for transfection into cells. To generate effective sgRNA sequences, a variety of online resources are available.

Structure

High degree of accuracy and very straightforward construction are provided by CRISPR-Cas9. It is dependent on the target sequence and the protospacer adjacent motif (PAM) sequence for its specificity. Each CRISPR locus's target sequence in the crRNA array is 20 bases long. Typical crRNA arrays have a variety of distinct target sequences. Cas9 proteins select the appropriate location on the host's genome by utilising the sequence to create bonds with base pairs on the host DNA. The sequence can be altered and independently produced because it is not a component of the Cas9 protein.

Cas9 recognises the PAM sequence on the host genome. It is difficult to change Cas9 to recognise a different PAM sequence. The SpCas9 PAM sequence, for example, is 5'-NGG-3' and occurs around every 8 to 12 base pairs in the human genome, so this is ultimately not too restrictive. It is also often a fairly brief and generic pattern that occurs repeatedly at numerous locations throughout the genome.

The Cas9 protein locates the correct sequence in the host cell's DNA with the help of the crRNA after being joined into a plasmid and transfected into cells. Depending on the Cas9 variant, the Cas9 protein then either creates a single-stranded break or a double-stranded break at the correct location in the DNA.

In response to correctly spaced single-stranded breaks, host DNA can undertake homology directed repair, which is less error-prone than the non-homologous end joining that often takes place after a double-stranded break. By supplying a DNA repair template, it is possible to insert a certain DNA sequence at a precise place inside the genome. The repair template should extend 40 to 90 base pairs past the Cas9-induced DNA break. The goal is for the cell's native HDR process, which will utilise the provided repair template, to incorporate the new sequence into the genome. This new code has now been integrated into the cell's genetic material and is transmitted to the cell's daughter cells.

Delivery

Viral and non-viral techniques can be used to deliver Cas9, sgRNA, and related complexes into cells. It is a common approach to electroporate DNA, RNA, or rib nucleocomplexes, although it can have negative consequences on the target cells. The introduction of sgRNAs in association with Cas9 into cells has also been accomplished chemically, using transfection techniques based on lipids and peptides. Transfection has also been accomplished using delivery based on nanoparticles. Cell types that are more difficult to transfect, such as stem cells, neurons, and hematopoietic cells, call for more efficient delivery strategies, such as those based on lentivirus (LVs), adenovirus (AdV), and adeno-associated virus (AAV).

When various system components, such as the full CRISPR/Cas9 structure to Cas9-gRNA complexes, are provided in assembled form as opposed to employing transgenics, it has been observed that the efficiency of CRISPR-Cas9 is significantly increased. This has found particular value in the broad commercialization of genetically engineered crops. Since the host's replication machinery is not required to make these proteins, there is essentially no likelihood that the sgRNA will recognise its sequence, reducing the possibility of off-target consequences.

Controlled Genome Editing

The CRISPR-Cas9 system has continued to be developed with the goal of enhancing use control. Research is being done to specifically increase the specificity, effectiveness, and granularity of this system's editing power. The part of the system that a technique modifies allows for further categorization and division of techniques. These include altering the sgRNA, creating novel Cas protein variations, utilising an entirely different effector protein, or applying an algorithmic technique to find already-existing ideal solutions.

The CRISPR-Cas9 system has to be more precise because its off-target effects can have detrimental impacts on a cell's genome and should be used with caution. Maximising system safety therefore means minimising off-target consequences. Effector proteins with efficiency and specificity comparable to the original SpCas9 that can target the formerly unmarketable sequences and a variant with almost no off-target mutations are examples of novel Cas9 protein modifications that boost specificity. Additionally, research has been done on creating novel Cas9 proteins, some of which partially replace the RNA nucleotides in crRNA with DNA and others that use a structure-guided method to create Cas9 mutants with fewer off-target effects. It has been demonstrated that highly stabilised gRNAs and sgRNAs that have been shortened iteratively also reduce off-target effects. To forecast the affinity of and develop distinctive sequences for the system to maximise specificity for targets, computational techniques such as machine learning have been applied.

Several CRISPR-Cas9 variants enable genome editing or gene activation using an external trigger, such as light or tiny chemicals. These include photoactivatable CRISPR systems developed by fusing light-responsive protein partners with an activator domain and a dCas9 to activate genes, or by fusing related light-responsive domains with two constructs of split-Cas9, or by modifying Cas9 with caged unnatural amino acids, or by modifying guide RNAs with photocleavable complements to modify the genome.

Genome editing with small molecules can be regulated using intein-linked Cas9s that are 4-HT responsive, allosteric Cas9s that activate binding and cleavage when 4-hydroxytamoxifen (4-HT) is added, or Cas9s that are 4-HT responsive when fused to four ERT2 domains.

Split-Cas9 systems that are activated by rapamycin and two constructs of split-Cas9 that have been fused with FRB and FKBP segments allow Cas9 fragments to dimerize. With the help of the small chemical doxycycline, Cas9 transcription has been successfully induced in other investigations. Additionally, small compounds can be employed to enhance homology-directed repair, frequently by blocking the pathway for non-homologous end joining. Genome editing with small molecules can be regulated using intein-linked Cas9s that are 4-HT responsive, allosteric Cas9s that activate binding and cleavage when 4-hydroxytamoxifen (4-HT) is added, or Cas9s that are 4-HT responsive when fused to four ERT2 domains. Split-Cas9 systems that are activated by rapamycin and two constructs of split-Cas9 that have been fused with FRB and FKBP segments allow Cas9 fragments to dimerize. With the help of the small chemical doxycycline, Cas9 transcription has been successfully induced in other investigations. Additionally, small compounds can be employed to enhance homology-directed repair, frequently by blocking the pathway for non-homologous end joining.

To specifically alter one or two bases in the target sequence, CRISPR also uses single base-pair editing proteins. It was combined with specialised enzymes that, at first, could only reverse C to T and G to A mutations. Eventually, this was completed without the need for DNA cleavage. The CRISPR-Cas9 method for base editing can also change C to G and its reverse by fusing with another enzyme.

CRISPR Screening

Anywhere that guide ribonucleic acids (gRNA) may connect with the protospacer adjacent motif (PAM) sequence, double-strand breaks (DSBs) can be brought about by the CRISPR/Cas9 system, a gene-editing technique. Cas9 active-site mutants, commonly referred to as Cas9 nickases, can also cause single-strand nicks. The Cas9-endonuclease can be delivered to a gene of interest and produce DSBs by only altering the sequence of gRNA. The invention of CRISPR-knockout (KO) libraries for mouse and human cells, which can either cover specific gene sets of interest or the entire genome, was made possible by the efficacy of Cas9-endonuclease and the simplicity with which genes can be targeted. Scientists can produce a high-throughput, systematic genetic disruption in living model organisms thanks to CRISPR screening. For a complete understanding of gene function and epigenetic regulation, this genetic disruption is required. Pooled CRISPR libraries have the benefit of enabling simultaneous targeting of more genes.

Knock-out libraries contain an antibiotic or fluorescent selection marker that may be used to identify transduced cells and are designed to achieve equitable representation and performance across all expressed gRNAs. Two plasmid systems can be found in CRISPR/Cas9 libraries. The first is an all-in-one plasmid, in which a transfected cell simultaneously produces sgRNA and Cas9. The second approach uses two vectors and independent delivery of the Cas9 and sgRNA plasmids. It is crucial to use viral transduction to deliver thousands of different sgRNA-containing vectors to a single vessel of cells at low multiplicity of infection; this reduces the likelihood that a single cell clone will receive more than one type of sgRNA and prevents incorrect genotype-phenotype assignment.

In order to determine the quantity of sgRNAs, it is necessary to do deep sequencing (NGS, next generation sequencing) of PCR-amplifed plasmid DNA. The library can consequently infect cells of interest, which can subsequently be chosen based on phenotypic. Positive and negative selection come in two flavours. Negative selection effectively detects dead or slowly developing cells. It can pinpoint genes that are necessary for survival, which can then be used to find potential molecular targets for medications. On the other hand, positive selection results in a population of randomly mutated populations that have a growth advantage. Following selection, NGS is used to harvest and sequence genomic DNA. The target gene that each sgRNA relates to is annotated, and depletion or enrichment of the sgRNAs is identified and compared to the original sgRNA library. The genes that are significantly more likely to be relevant to the desired phenotype are subsequently found by statistical analysis.

Applications

Disease Models

In the world of genetics, Cas9 genome editing has made it possible to produce transgenic models quickly and effectively. To simulate the spread of illnesses and the cell's reaction to and defence against infection, Cas9 can be simply introduced into the target cells coupled with sgRNA using plasmid transfection. Cas9's ability to be introduced in vivo makes it possible to create models of gene function and mutation consequences that are more accurate while also preventing the off-target mutations that are frequently seen with earlier techniques for genetic engineering.

The genomic modelling revolution brought about by CRISPR and Cas9 is not limited to mammals. The introduction of Cas9 has improved the resolution of conventional genomic models, such as Drosophila melanogaster, one of the original model species. Cas9 makes use of cell-specific promoters to enable regulated Cas9 use. Because the Cas9 enzyme only affects specific cell types, it offers a precise way for treating disorders. To enhance the benefits of the therapy, the cells undergoing Cas9 treatment can also be taken out and put back in.

At any stage of an organism's development, CRISPR-Cas9 can be used to edit the DNA of living things in vivo and remove specific genes or even entire chromosomes.

The human chromosomes 14 and 21, in embryonic stem cell lines and aneuploid mice, respectively, as well as the Y and X chromosomes of adult lab mice, have all been successfully removed in vivo utilising CRISPR technology. This approach may be helpful for addressing genetic conditions like Down syndrome and intersex diseases that are brought on by aberrant numbers of chromosomes.

Numerous model species, including Escherichia coli, Saccharomyces cerevisiae, Candida albicans, Methanosarcina acetivorans, Caenorhabditis elegans, Arabidopsis spp, Danio rerio, and Mus musculus, have successfully used CRISPR-Cas9 to modify the genome in vivo. Basic biological research, the development of illness models, and the experimental treatment of disease models have all had success.

Off-target effects (editing of genes other than the ones targeted) have generated concerns that they could skew the outcomes of a CRISPR gene editing experiment (i.e., the reported phenotypic change might not be caused by changing the target gene, but rather some other gene). CRISPR has undergone modifications to reduce the likelihood of off-target consequences. It's frequently advised to do orthogonal CRISPR experiments to validate the outcomes of gene-editing studies.

Genetically edited creatures that imitate sickness or demonstrate what occurs when a gene is knocked down or altered are made easier to make thanks to CRISPR. CRISPR can be employed in non-germline cells to make local modifications that only affect specific cell populations within the organism, or it can be used at the germline level to generate animals in which the targeted gene is modified everywhere (i.e., in all cells, tissues, and organs of a multicellular organism).

CRISPR can be used to develop disease-related human cellular models. For instance, using human pluripotent stem cells, CRISPR has been used to specifically induce mutations in genes associated with FSGS and PKD, or polycystic kidney disease. The human kidney organoids produced from these CRISPR-modified pluripotent stem cells displayed disease-specific characteristics. Large, transparent cyst formations were created from kidney tubules by kidney organoids made from stem cells with PKD mutations. The cysts had the potential to grow to macroscopic sizes up to one centimetre in diameter. The filtering cells involved in FSGS, podocytes, were found to have junctional abnormalities in kidney organoids with mutations in a gene associated with the condition. This was related to the inability of podocytes to produce microvilli between neighbouring cells. Importantly, control organoids with comparable genetic backgrounds but without the CRISPR changes lacked these disease characteristics.

Modelling the long QT syndrome in cardiomyocytes created from pluripotent stem cells was done in a manner akin to that. These CRISPR-created cellular models with isogenic controls offer a fresh approach to researching human disease and evaluating medications.

Biomedicine

Many human diseases, particularly those with a genetic basis, have been suggested as candidates for treatment using CRISPR-Cas technology. It is a tool with the potential to correct mutations that cause disease because of its capacity to alter DNA sequences. Early studies using animal models indicate the potential of CRISPR-based therapies to treat a variety of illnesses, including cancer, progeria, beta-thalassemia, sickle cell disease, haemophilia, cystic fibrosis, Duchenne's muscular dystrophy, Huntington's disease, transthyretin amyloidosis, and heart disease. Additionally, CRISPR has been used to treat mosquitoes for malaria, which could eradicate the disease's vector and human victims.

Additionally, CRISPR may have uses in tissue engineering and regenerative medicine, such as the development of human blood arteries devoid of the MHC class II proteins that frequently result in transplant rejection. Additionally, CRISPR-Cas9 technology has showed promise in clinical trials treating beta thalassemia and sickle cell disease in human patients. However, there are still several restrictions on the technology's application to gene therapy: the necessity for a PAM sequence close to the target location, p53-mediated apoptosis caused by double-strand breaks caused by CRISPR, and immunogenic toxicity because the delivery mechanism is often a virus.