what are the applications of crispr

CRISPR/Cas9 Applications

• Basic Science. To date, the CRISPR/Cas9 gene editing tool appears to work in nearly every organism, from Caenorhabditis elegans to monkeys, and in every cell type: kidney, heart and those, ...
• Agriculture. ...
• Disease Modeling. ...
• Gene Therapy. ...

Full Answer

What are the most interesting uses of CRISPR?

Applications of CRISPR. Using CRISPR for genome editing. Using CRISPR libraries for screening. CRISPR/Cas9-mediated chromatin immunoprecipitation. Transcriptional activation and repression. Epigenetic editing with CRISPR/Cas9. Live …

What are some new applications of CRISPR?

Aug 07, 2020 · The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins has expanded the applications of genetic research in thousands of laboratories across the globe and is …

Who should be allowed to use CRISPR?

Aug 21, 2021 · The CRISPR/Cas-9 genome-editing tool has a wide number of applications in many areas including medicine, agriculture, and biotechnology. In agriculture, it could help in the design of new grains to improve their nutritional value.

What can CRISPR be used for?

Compared to the current applications of CRISPR technology in medical fields such as oncology, neuroscience or developmental biology, there is a limited number of applications in the transplantation. However, promising improvements are continually developing, particularly those related to xenotransplantation.

What is CRISPR in gene therapy?

The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins has expanded the applications of genetic research in thousands of laboratories across the globe and is redefining our approach to gene therapy. Traditional gene therapy has raised some concerns, as its reliance on viral vector delivery of therapeutic transgenes can cause both insertional oncogenesis and immunogenic toxicity. While viral vectors remain a key delivery vehicle, CRISPR technology provides a relatively simple and efficient alternative for site-specific gene editing, obliviating some concerns raised by traditional gene therapy. Although it has apparent advantages, CRISPR/Cas9 brings its own set of limitations which must be addressed for safe and efficient clinical translation. This review focuses on the evolution of gene therapy and the role of CRISPR in shifting the gene therapy paradigm. We review the emerging data of recent gene therapy trials and consider the best strategy to move forward with this powerful but still relatively new technology.

What is the purpose of CRISPR/CAS9?

CRISPR/Cas9 is a simple two-component system used for effective targeted gene editing. The first component is the single-effector Cas9 protein, which contains the endonuclease domains RuvC and HNH. RuvC cleaves the DNA strand non-complementary to the spacer sequence and HNH cleaves the complementary strand. Together, these domains generate double-stranded breaks (DSBs) in the target DNA. The second component of effective targeted gene editing is a single guide RNA (sgRNA) carrying a scaffold sequence which enables its anchoring to Cas9 and a 20 base pair spacer sequence complementary to the target gene and adjacent to the PAM sequence. This sgRNA guides the CRISPR/Cas9 complex to its intended genomic location. The editing system then relies on either of two endogenous DNA repair pathways: non-homologous end-joining (NHEJ) or homology-directed repair (HDR) ( Figure 2 ). NHEJ occurs much more frequently in most cell types and involves random insertion and deletion of base pairs, or indels, at the cut site. This error-prone mechanism usually results in frameshift mutations, often creating a premature stop codon and/or a non-functional polypeptide. This pathway has been particularly useful in genetic knock-out experiments and functional genomic CRISPR screens, but it can also be useful in the clinic in the context where gene disruption provides a therapeutic opportunity. The other pathway, which is especially appealing to exploit for clinical purposes, is the error-free HDR pathway. This pathway involves using the homologous region of the unedited DNA strand as a template to correct the damaged DNA, resulting in error-free repair. Experimentally, this pathway can be exploited by providing an exogenous donor template with the CRISPR/Cas9 machinery to facilitate the desired edit into the genome ( 30 ).

What is Cas9 in CRISPR?

Precise Gene Editing. (A) CRISPR/Cas9-HDR. Cas9 induces a DSB. The exogenous ssODN carrying the sequence for the desired edit and homology arms is used as a template for HDR-mediated gene modification. (B) Base Editor. dCas9 or Cas9n is tethered to the catalytic portion of a deaminase. Cytosine deaminase catalyzes the formation of uridine from cytosine. DNA mismatch repair mechanisms or DNA replication yield an C:G to T:A single nucleotide base edit. Adenosine deaminase catalyzes the formation of inosine from adenosine. DNA mismatch repair mechanisms or DNA replication yield an A:T to G:C single nucleotide base edit. (C) Prime Editor. Cas9n is tethered to the catalytic portion of reverse transcriptase. The prime editor system uses pegRNA, which contains the guide spacer sequence, reverse transcriptase primer, which includes the sequence for the desired edit and a primer binding site (PBS). PBS hybridizes with the complementary region of the DNA and reverse transcriptase transcribes new DNA carrying the desired edit. After cleavage of the resultant 5′ flap and ligation, DNA repair mechanisms correct the unedited strand to match the edited strand. HDR, homology directed repair. DSB, double stranded break; SSB, single-stranded break; ssODN, single-stranded oligodeoxynucleotide.

What is the CRISPR locus?

The bacterial CRISPR locus was first described by Francisco Mojica ( 23) and later identified as a key element in the adaptive immune system in prokaryotes ( 24 ). The locus consists of snippets of viral or plasmid DNA that previously infected the microbe (later termed “spacers”), which were found between an array of short palindromic repeat sequences. Later, Alexander Bolotin discovered the Cas9 protein in Streptococcus thermophilus, which unlike other known Cas genes, Cas9 was a large gene that encoded for a single-effector protein with nuclease activity ( 25 ). They further noted a common sequence in the target DNA adjacent to the spacer, later known as the protospacer adjacent motif (PAM)—the sequence needed for Cas9 to recognize and bind its target DNA ( 25 ). Later studies reported that spacers were transcribed to CRISPR RNAs (crRNAs) that guide the Cas proteins to the target site of DNA ( 26 ). Following studies discovered the trans-activating CRISPR RNA (tracrRNA), which forms a duplex with crRNA that together guide Cas9 to its target DNA ( 27 ). The potential use of this system was simplified by introducing a synthetic combined crRNA and tracrRNA construct called a single-guide RNA (sgRNA) ( 28 ). This was followed by studies demonstrating successful genome editing by CRISPR/Cas9 in mammalian cells, thereby opening the possibility of implementing CRISPR/Cas9 in gene therapy ( 29) ( Figure 1 ).

What are the two types of CRISPR editors?

Currently, the two types of CRISPR base editors are cytidine base editors (CBEs) and adenosine base editors (ABEs). CBEs catalyze the conversion of cytidine to uridine, which becomes thymine after DNA replication. ABEs catalyze the conversion of adenosine to inosine which becomes guanine after DNA replication ( 87 ).

Does CRISPR cause apoptosis?

CRISPR- induced DSBs often trigger apoptosis rather than the intended gene edit ( 68 ). Further safety concerns were revealed when using this tool in human pluripotent stem cells (hPSCs) which demonstrated that p53 activation in response to the toxic DSBs introduced by CRISPR often triggers subsequent apoptosis ( 69 ). Thus, successful CRISPR edits are more likely to occur in p53 suppressed cells, resulting in a bias toward selection for oncogenic cell survival ( 70 ). In addition, large deletions spanning kilobases and complex rearrangements as unintended consequences of on-target activity have been reported in several instances ( 71, 72 ), highlighting a major safety issue for clinical applications of DSB-inducing CRISPR therapy. Other variations of Cas9, such as catalytically inactive endonuclease dead Cas9 (dCas9) in which the nuclease domains are deactivated, may provide therapeutic utility while mitigating the risks of DSBs ( 73 ). dCas9 can transiently manipulate expression of specific genes without introducing DSBs through fusion of transcriptional activating or repressing domains or proteins to the DNA-binding effector ( 74 ). Other variants such as Cas9n can also be considered, which induces SSBs rather than DSBs. Further modifications of these Cas9 variants has led to the development of base editors and prime editors, a key innovation for safe therapeutic application of CRISPR technology (see Precision Gene Editing With CRISPR section).

Is somatic editing allowed in CRISPR?

While somatic editing for CRISPR therapy has been permitted after careful consideration, human germline editing for therapeutic intent remains highly controversial. With somatic edition, any potential risk would be contained within the individual after informed consent to partake in the therapy. Embryonic editing not only removes autonomy in the decision-making process of the later born individuals, but also allows unforeseen and permanent side effects to pass down through generations. This very power warrants proceeding with caution to prevent major setbacks as witnessed by traditional gene therapy. However, a controversial CRISPR trial in human embryos led by Jiankui He may have already breached the ethical standards set in place for such trials. This pilot study involved genetic engineering of the C-C chemokine receptor type 5 ( CCR5) gene in human embryos, with the intention of conferring HIV-resistance, as seen by a naturally occurring CCR5 Δ 32 mutation in a few individuals ( 108 ). However, based on the limited evidence, CRISPR/Cas9 was likely used to target this gene, but rather than replicate the naturally observed and beneficial 32-base deletion, the edits merely induced DSBs at one end of the deletion, allowing NHEJ to repair the damaged DNA while introducing random, uncharacterized mutations. Thus, it is unknown whether the resultant protein will function similarly to the naturally occurring CCR5 Δ 32 protein and confer HIV resistance. In addition, only one of the two embryos, termed with the pseudonym Nana, had successful edits in both copies of the CCR5 gene, whereas the other embryo, with pseudonym Lulu, had successful editing in only one copy. Despite these findings, both embryos were implanted back into their mother, knowing that the HIV-resistance will be questionable in Nana and non-existent in Lulu ( 109, 110 ).

Why is CRISPR used in gene editing?

Due to ease of use and higher editing efficiency, CRISPR/Cas9 became a popular gene-editing tool to edit the genome of different cell types (cell lines, primary cells, iPSC) and to generate transgenic animals in a short amount of time20, 21.

Which animal is used for xenotransplantation?

Chimpanzees and baboons have been used in initial trials, but in the modern era of xenotransplantation, porcine are currently preferred due to their similar organ size, ease of breeding, high offspring number, and less disease transmission risk compared to NHP.

What is the purpose of CRISPR in UPenn?

The UPenn trial aims to use CRISPR to perform three different genetic modifications on T-cells from patients with myeloma, sarcoma or melanoma, using CRISPR not only to disrupt PD-1 receptor expression, but also to knock out portions of the T-cell’s primary receptor.

How has personalised medicine been made possible?

The advent of personalised medicine has been made possible by inexpensive genome sequencing technology and ever-more sophisticated biochemical tools. These have fuelled efforts to create targeted therapies specifically tailored to cater for the genetic identity of a particular patient, or to target specific genetic variants of a condition or disease.

Is CRISPR the only gene editing technique?

CRISPR is not the only or even the first technique developed for gene manipulation/editing – older technologies employing Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have been known, and have been the subject of much interest and investment, for years.

What is the DNA recorder?

The tool acts as a recorder of events in the lifetime of a cell, such as exposure to antibiotics, nutrients, viruses and light.

What is the company that edits the genome of race horses?

The Argentinian company Kheiron-Biotech is editing the genome of race horses to make breeds that are faster, stronger and better jumpers. Thanks to CRISPR, researchers at the company were able to modify the gene encoding for myostatin, a protein that is crucial for the growth of muscles.

How does gene editing help algae?

Gene editing could improve the production of biofuels by algae. Using CRISPR-Cas9 , the company Synthetic Genomics has created strains of algae that produce twice as much fat, which is then used to produce biodiesel. In particular, the gene editing tool allowed scientists to find and remove genes that limit the production of fats.

What is gene editing in dogs?

The gene editing tool has been proposed as a way of removing the genetic diseases that abound in pure breed dogs. A great example are Dalmatians, which often carry a genetic mutation that makes them prone to suffer from bladder stones.

Is CRISPR a good tool for treating diseases?

Furthermore, clinical trials using the gene editing tool to treat diseases ranging from cancer to blindness and AIDS are underway.

Can CRISPR be used for celiacs?

Another research group, in the Netherlands, is using CRISPR-Cas9 to modify the DNA of wheat to remove gluten , making it suitable for celiacs. However, the EU’s strict regulation on using CRISPR gene editing in plants might make it difficult for this project to see the light, at least in Europe.

Can you make peanuts with CRISPR?

With CRISPR, it could be possible to make milk, eggs or peanuts that are safe for everyone to eat. “There are four proteins within egg white that cause allergy,” Tim Doran, researcher at Australia’s CSIRO, explained in a podcast. “We’re essentially rewriting those regions of the gene that are recognised by the immune system and cause an allergic reaction.”

What is CRISPR CAS?

CRISPR-Cas is a technique that allows the quick and easy alteration of genes. This technique has undergone a rapid development. This leads to the question: what do we want to achieve with CRISPR-Cas?

Which class of scissors are able to modify DNA strands?

This led to the discovery of a class of simple scissors – CRISPR-Cas9 – that have been revealed able to modify DNA-strands in many different kinds of organisms, including micro-organisms, plants, animals and people.

What is the mechanism of DNA scissors?

Protein scissors, a natural defence mechanism. The DNA scissors they discovered is a great example of an ingenious natural defence mechanism. The E. coli bacteria’s defence mechanism is able to recognise and disarm the DNA of viruses that invade the bacterium by cutting it. CRIPSR-Cas is known as the cutting mechanism.

Abstract

Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string.

Introduction

The development of recombinant DNA technology in the 1970s marked the beginning of a new era for biology. For the first time, molecular biologists gained the ability to manipulate DNA molecules, making it possible to study genes and harness them to develop novel medicine and biotechnology.

Programmable Nucleases as Tools for Efficient and Precise Genome Editing

A series of studies by Haber and Jasin ( Rudin et al., 1989; Plessis et al., 1992; Rouet et al., 1994; Choulika et al., 1995; Bibikova et al., 2001; Bibikova et al., 2003) led to the realization that targeted DNA double-strand breaks (DSBs) could greatly stimulate genome editing through HR-mediated recombination events.

CRISPR-Cas9: From Yogurt to Genome Editing

The recent development of the Cas9 endonuclease for genome editing draws upon more than a decade of basic research into understanding the biological function of the mysterious repetitive elements now known as CRISPR ( Figure 3 ), which are found throughout the bacterial and archaeal diversity.

Structural Organization and Domain Architecture of Cas9

The family of Cas9 proteins is characterized by two signature nuclease domains, RuvC and HNH, each named based on homology to known nuclease domain structures ( Figure 2C ).

Metagenomic, Structural, and Functional Diversity of Cas9

Cas9 is exclusively associated with the type II CRISPR locus and serves as the signature type II gene. Based on the diversity of associated Cas genes, type IICRISPR loci are further subdivided into three subtypes (IIA–IIC) ( Figure 5B) ( Makarova et al., 2011a; Chylinski et al., 2013 ).

Protospacer Adjacent Motif: Cas9 Target Range and Search Mechanism

A critical feature of the Cas9 system is the protospacer-adjacent motif (PAM), which flanks the 3′ end of the DNA target site ( Figure 2C) and dictates the DNA target search mechanism of Cas9.