crispr gene editing


  • [119] The efficiency of Cas9-endonuclease and the ease by which genes can be targeted led to the development of CRISPR-knockout (KO) libraries both for mouse and human cells,
    which can cover either specific gene sets of interest or the whole-genome.

  • CRISPRs are much easier to design because the process requires synthesizing only a short RNA sequence, a procedure that is already widely used for many other molecular biology
    techniques (e.g.

  • [86][87] Research has also been conducted in engineering new Cas9 proteins, including some that partially replace RNA nucleotides in crRNA with DNA and a structure-guided
    Cas9 mutant generating procedure that all had reduced off-target effects.

  • Spatiotemporal control is a form of removing off-target effects—only certain cells or parts of the organism may need to be modified, and thus light or small molecules can
    be used as a way to conduct this.

  • [12] History Other methods[edit] In the early 2000s, German researchers began developing zinc finger nucleases (ZFNs), synthetic proteins whose DNA-binding domains enable
    them to create double-stranded breaks in DNA at specific points.

  • [151] These CRISPR-generated cellular models, with isogenic controls, provide a new way to study human disease and test drugs.

  • The repair template is also uniquely designed for each application, as it must complement to some degree the DNA sequences on either side of the cut and also contain whatever
    sequence is desired for insertion into the host genome.

  • [127] The ability of Cas9 to be introduced in vivo allows for the creation of more accurate models of gene function and mutation effects, all while avoiding the off-target
    mutations typically observed with older methods of genetic engineering.

  • By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell’s genome can be cut at a desired location, allowing existing genes to be
    removed and/or new ones added in vivo.

  • [8] The ease with which researchers can insert Cas9 and template RNA in order to silence or cause point mutations at specific loci has proved invaluable to the quick and efficient
    mapping of genomic models and biological processes associated with various genes in a variety of eukaryotes.

  • [122] It is important to deliver thousands of unique sgRNAs-containing vectors to a single vessel of cells by viral transduction at low multiplicity of infection (MOI, typically
    at 0.1-0.6), it prevents the probability that an individual cell clone will get more than one type of sgRNA otherwise it can lead to incorrect assignment of genotype to phenotype.

  • [172] Cancer[edit] CRISPR has also found many applications in developing cell-based immunotherapies.

  • [168][169] Nevertheless, there remains a few limitations of the technology’s use in gene therapy: the relatively high frequency of off-target effect, the requirement for a
    PAM sequence near the target site, p53 mediated apoptosis by CRISPR-induced double-strand breaks and immunogenic toxicity due to the delivery system typically by virus.

  • [129] Concerns have been raised that off-target effects (editing of genes besides the ones intended) may confound the results of a CRISPR gene editing experiment (i.e.

  • [120] Apart from knock-out there are also knock-down (CRISPRi) and activation (CRISPRa) libraries, which using the ability of proteolytically deactivated Cas9-fusion proteins
    (dCas9) to bind target DNA, which means that gene of interest is not cut but is over-expressed or repressed.

  • [128] CRISPR-Cas9 can be used to edit the DNA of organisms in vivo and to eliminate individual genes or even entire chromosomes from an organism at any point in its development.

  • Cas9 can be easily introduced into the target cells along with sgRNA via plasmid transfection in order to model the spread of diseases and the cell’s response to and defense
    against infection.

  • Specificity is an important aspect to improve the CRISPR-Cas9 system because the off-target effects it generates have serious consequences for the genome of the cell and invokes
    caution for its use.

  • [166] CRISPR may also have applications in tissue engineering and regenerative medicine, such as by creating human blood vessels that lack expression of MHC class II proteins,
    which often cause transplant rejection.

  • Knock-out libraries are created in a way to achieve equal representation and performance across all expressed gRNAs and carry an antibiotic or fluorescent selection marker
    that can be used to recover transduced cells.

  • [5][6][7] Working like genetic scissors, the Cas9 nuclease opens both strands of the targeted sequence of DNA to introduce the modification by one of two methods.

  • CRISPR may be used at the germline level to create organisms in which the targeted gene is changed everywhere (i.e.

  • The crRNA is uniquely designed for each application, as this is the sequence that Cas9 uses to identify and directly bind to specific sequences within the host cell’s DNA.

  • [84][85] Since the host’s replication machinery is not needed to produce these proteins, the chance of the recognizing sequence of the sgRNA is almost none, decreasing the
    chance of off-target effects.

  • [154] In 2016, the United States Food and Drug Administration (FDA) approved a clinical trial in which CRISPR would be used to alter T cells extracted from people with different
    kinds of cancer and then administer those engineered T cells back to the same people.

  • Once incorporated, this new sequence is now part of the cell’s genetic material and passes into its daughter cells.

  • One issue with this approach is that it requires the removal of the HIV genome from almost all cells, which can be difficult to realistically achieve.

  • [55] Once these sequences have been assembled into a plasmid and transfected into cells, the Cas9 protein with the help of the crRNA finds the correct sequence in the host
    cell’s DNA and – depending on the Cas9 variant – creates a single- or double-stranded break at the appropriate location in the DNA.

  • [120][121] CRISPR screening helps scientist to create a systematic and high-throughput genetic perturbation within live model organisms.

  • While genome editing in eukaryotic cells has been possible using various methods since the 1980s, the methods employed had proved to be inefficient and impractical to implement
    on a large scale.

  • [92][93] Several variants of CRISPR-Cas9 allow gene activation or genome editing with an external trigger such as light or small molecules.

  • [14] CRISPR can also target several DNA sites simultaneously simply by introducing different gRNAs.

  • [55] The goal is for the cell’s native HDR process to utilize the provided repair template and thereby incorporate the new sequence into the genome.

  • [79][80][81] Efficiency of CRISPR-Cas9 has been found to greatly increase when various components of the system including the entire CRISPR/Cas9 structure to Cas9-gRNA complexes
    delivered in assembled form rather than using transgenics.

  • Electroporation of DNA, RNA, or ribonucleocomplexes is a common technique, though it can result in harmful effects on the target cells.

  • Therefore, genomic engineering by CRISPR-Cas9 gives researchers the ability to generate targeted random gene disruption.

  • Efficiency of the CRISPR-Cas9 system is also greatly increased by proper delivery of the DNA instructions for creating the proteins and necessary reagents.

  • [90][91] Computational methods including machine learning have been used to predict the affinity of and create unique sequences for the system to maximize specificity for
    given targets.

  • Delivery[edit] See also: Transfection Delivery of Cas9, sgRNA, and associated complexes into cells can occur via viral and non-viral systems.

  • Both zinc finger nucleases and TALENs require the design and creation of a custom protein for each targeted DNA sequence, which is a much more difficult and time-consuming
    process than that of designing guide RNAs.

  • Biomedicine[edit] CRISPR-Cas technology has been proposed as a treatment for multiple human diseases, especially those with a genetic cause.

  • However, its use in human germline genetic modification is highly controversial.

  • [39][5] In June 2021, the first, small clinical trial of intravenous CRISPR gene editing in humans concludes with promising results.

  • [107][108] Small molecules can also be used to improve homology directed repair,[109] often by inhibiting the non-homologous end joining pathway.

  • [152] Its ability to modify specific DNA sequences makes it a tool with potential to fix disease-causing mutations.

  • [124][125][126] Applications Disease models[edit] Cas9 genomic modification has allowed for the quick and efficient generation of transgenic models within the field of genetics.

  • Cas9 is an accurate method of treating diseases due to the targeting of the Cas9 enzyme only affecting certain cell types.

  • These include using a different variants or novel creations of the Cas protein, using an altogether different effector protein, modifying the sgRNA, or using an algorithmic
    approach to identify existing optimal solutions.

  • It made CRISPR/Cas9 system even more interesting in gene editing.

  • Novel variations of Cas9 proteins that increase specificity include effector proteins with comparable efficiency and specificity to the original SpCas9 that are able to target
    the previously untargetable sequences and a variant that has virtually no off-target mutations.

  • However, this is ultimately not too limiting, as it is typically a very short and nonspecific sequence that occurs frequently at many places throughout the genome (e.g.

  • These cells are sourced from healthy donors and are edited to attack cancer cells and avoid being seen as a threat by the recipient’s immune system, and then multiplied into
    huge batches which can be given to large numbers of recipients.

  • This technology thus represents a novel form of antimicrobial therapy and a strategy by which to manipulate bacterial populations.

  • While effective treatments exist which can allow patients to live healthy lives, HIV is retroactive meaning that it embeds an inactive version of itself in the human genome.

  • [57][58] Overview of the transfection and DNA cleaving by CRISPR-Cas9 (crRNA and tracrRNA are often joined as a single strand of RNA when designing a plasmid)[55] Structure[edit]
    CRISPR-Cas9 offers a high degree of fidelity and relatively simple construction.

  • [144][145][146] CRISPR can be utilized to create human cellular models of disease.

  • CRISPR is used to edit the cells in order to reduce the chance the patient’s body will reject the transplant.

  • Cas9 derived from the bacterial species Streptococcus pyogenes has facilitated targeted genomic modification in eukaryotic cells by allowing for a reliable method of creating
    a targeted break at a specific location as designated by the crRNA and tracrRNA guide strands.

  • [78] Controlled genome editing[edit] Further improvements and variants of the CRISPR-Cas9 system have focused on introducing more control into its use.

  • Orthogonal CRISPR experiments are often recommended to confirm the results of a gene editing experiment.

  • HDR employs the use of similar DNA sequences to drive the repair of the break via the incorporation of exogenous DNA to function as the repair template.

  • These end-joining pathways can often result in random deletions or insertions at the repair site, which may disrupt or alter gene functionality.

  • [111] CRISPR also utilizes single base-pair editing proteins to create specific edits at one or two bases in the target sequence.

  • [167] In addition, clinical trials to cure beta thalassemia and sickle cell disease in human patients using CRISPR-Cas9 technology have shown promising results.

  • in all cells/tissues/organs of a multicellular organism), or it may be used in non-germline cells to create local changes that only affect certain cell populations within
    the organism.

  • [9] CRISPR-Cas9 genome editing techniques have many potential applications, including in medicine and agriculture.

  • The cells undergoing the Cas9 therapy can also be removed and reintroduced to provide amplified effects of the therapy.


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