CRISPR gene editing
CRISPR gene editing (pronounced /ˈkrɪspər/ "crisper") is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. 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.[1]
The technique is considered highly significant in
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. Knock-in mutations, facilitated via homology directed repair (HDR), is the traditional pathway of targeted genomic editing approaches.[1] This allows for the introduction of targeted DNA damage and repair. 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.[1] This method relies on the periodic and isolated occurrence of DNA damage at the target site in order for the repair to commence. Knock-out mutations caused by CRISPR-Cas9 result from the repair of the double-stranded break by means of non-homologous end joining (NHEJ) or POLQ/polymerase theta-mediated end-joining (TMEJ). These end-joining pathways can often result in random deletions or insertions at the repair site, which may disrupt or alter gene functionality. Therefore, genomic engineering by CRISPR-Cas9 gives researchers the ability to generate targeted random gene disruption.
While genome editing in eukaryotic cells has been possible using various methods since the 1980s, the methods employed had proven to be inefficient and impractical to implement on a large scale. With the discovery of CRISPR and specifically the Cas9 nuclease molecule, efficient and highly selective editing became possible. 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.[8] The ease with which researchers can insert Cas9 and template RNA in order to silence or cause point mutations at specific loci has proven to be invaluable to the quick and efficient mapping of genomic models and biological processes associated with various genes in a variety of eukaryotes. Newly engineered variants of the Cas9 nuclease have been developed that significantly reduce off-target activity.[9]
CRISPR-Cas9 genome editing techniques have many potential applications. The use of the CRISPR-Cas9-gRNA complex for genome editing
History
Other methods
In the early 2000s, German researchers began developing
Whereas methods such as
Discovery
In 2005, Alexander Bolotin at the French National Institute for Agricultural Research (INRA) discovered a CRISPR locus that contained novel Cas genes, significantly one that encoded a large protein known as Cas9.[20]
In 2006, Eugene Koonin at the US National Center for Biotechnology information, NIH, proposed an explanation as to how CRISPR cascades as a bacterial immune system.[20]
In 2007, Philippe Horvath a Danisco France SAS displayed experimentally how CRISPR systems are an adaptive immune system, and integrate new phage DNA into the CRISPR array, which is how they fight off the next wave of attacking phage.[20]
In 2012, the research team led by the University of California, Berkeley, professor Jennifer Doudna and Umea University professor Emmanuelle Charpentier, were the first people to identify, disclose, and file a patent application for the CRISPR-Cas9 system needed to edit DNA.[20] They also published their finding that CRISPR-Cas9 could be programmed with RNA to edit genomic DNA, now considered one of the most significant discoveries in the history of biology.
Patents and commercialization
As of November 2013[update], SAGE Labs (part of Horizon Discovery group) had exclusive rights from one of those companies to produce and sell genetically engineered rats and non-exclusive rights for mouse and rabbit models.[21] By 2015[update], Thermo Fisher Scientific had licensed intellectual property from ToolGen to develop CRISPR reagent kits.[22]
As of December 2014[update],
The first set of patents was awarded to the Broad team in 2015, prompting attorneys for the CVC group to request the first interference proceeding.
Shortly after, University of California filed an appeal of this ruling.[31][32] In 2019 the second interference dispute was opened. This was in response to patent applications made by CVC that required the appeals board to determine the original inventor of the technology. The USPTO ruled in March 2022 against UC, stating that the Broad Institute were first to file. The decision affected many of the licensing agreements for the CRISPR editing technology that was licensed from UC Berkeley. UC stated its intent to appeal the USPTO's ruling.[33]
Recent events
In March 2017, the European Patent Office (EPO) announced its intention to allow claims for editing all types of cells to Max-Planck Institute in Berlin, University of California, and University of Vienna,[34][35] and in August 2017, the EPO announced its intention to allow CRISPR claims in a patent application that MilliporeSigma had filed.[34] As of August 2017[update] the patent situation in Europe was complex, with MilliporeSigma, ToolGen, Vilnius University, and Harvard contending for claims, along with University of California and Broad.[36]
In July 2018, the
In February 2020, a US trial showed safe CRISPR gene editing on three cancer patients.[38]
In October 2020, researchers Emmanuelle Charpentier and Jennifer Doudna were awarded the Nobel Prize in Chemistry for their work in this field.[39][40] They made history as the first two women to share this award without a male contributor.[41][5]
In June 2021, the first, small clinical trial of intravenous CRISPR gene editing in humans concludes with promising results.[42][43]
In September 2021, the first CRISPR-edited food has gone on public sale in Japan. Tomatoes
In December 2021, it was reported that the first CRISPR-gene-edited marine animal/
A 2022 study has found that knowing more about CRISPR tomatoes had a strong effect on the participants' preference. "Almost half of the 32 participants from Germany who are scientists demonstrated constant choices, while the majority showed increased willingness to buy CRISPR tomatoes, mostly non-scientists."[50][51]
The UC Berkeley announced in May 2021 their intent to auction
In November 2023, the United Kingdom's
In December 2023, the FDA approved the first gene therapy in the US to treat patients with Sickle Cell Disease (SCD). The FDA approved two milestone treatments, Casgevy and Lyfgenia, representing the first cell-based gene therapies for the treatment of SCD.[57]
Genome engineering
CRISPR-Cas9 genome editing is carried out with a Type II CRISPR system. When utilized for genome editing, this system includes a ribonucleoprotein (RNP), consisting of Cas9, crRNA, and tracrRNA, along with an optional DNA repair template.
Major components
Component | Function |
---|---|
crRNA | Contains the guide RNA that locates the correct segment of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form), forming an active complex. |
tracrRNA | Binds to crRNA and forms an active complex. |
sgRNA | Single-guide RNAs are a combined RNA consisting of a tracrRNA and at least one crRNA. |
Cas9 (most commonly) | An enzyme whose active form is able to modify DNA. Many variants exist with different functions (i.e. single-strand nicking, double-strand breaking, DNA binding) due to each enzyme's DNA site recognition function. |
Repair template | DNA molecule used as a template in the host cell's DNA repair process, allowing insertion of a specific DNA sequence into the host segment broken by Cas9. |
Multiple crRNAs and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA).[59] This sgRNA can be included alongside the gene that codes for the Cas9 protein and made into a plasmid in order to be transfected into cells. Many online tools are available to aid in designing effective sgRNA sequences.[60][61]
Alternatives to Cas9
This section needs expansion. You can help by adding to it. (October 2021) |
Alternative proteins to Cas9 include the following:
Protein | Main use / characteristics | Year/s |
---|---|---|
Cas12 | ||
Cas13 | for RNA editing[64] | |
Cas3[65][66] | Creates a single-stranded wide gap[67] | 2019 |
CasMINI | About twice as compact as the more commonly used Cas9 and Cas12a.[68][69] | 2021 |
SuperFi-Cas9 | More accurate without a slow down in speed[70][71] | 2022 |
Cas7-11 | RNA editing[72] | 2022 |
Chromosome-templated DNA repair | Such a method is only applicable to organisms whose matching chromosome has the desired gene/s.
|
2022 |
Structure
CRISPR-Cas9 offers a high degree of fidelity and relatively simple construction. It depends on two factors for its specificity: the target sequence and the protospacer adjacent motif (PAM) sequence. The target sequence is 20 bases long as part of each CRISPR locus in the crRNA array.[58] A typical crRNA array has multiple unique target sequences. Cas9 proteins select the correct location on the host's genome by utilizing the sequence to bond with base pairs on the host DNA. The sequence is not part of the Cas9 protein and as a result is customizable and can be independently synthesized.[75][76]
The PAM sequence on the host genome is recognized by Cas9. Cas9 cannot be easily modified to recognize a different PAM sequence. 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. the SpCas9 PAM sequence is 5'-NGG-3' and in the human genome occurs roughly every 8 to 12 base pairs).[58]
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.[77]
Properly spaced single-stranded breaks in the host DNA can trigger homology directed repair, which is less error-prone than the non-homologous end joining that typically follows a double-stranded break. Providing a DNA repair template allows for the insertion of a specific DNA sequence at an exact location within the genome. The repair template should extend 40 to 90 base pairs beyond the Cas9-induced DNA break.[58] 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. Once incorporated, this new sequence is now part of the cell's genetic material and passes into its daughter cells. Combined transient inhibition of NHEJ and TMEJ by a small molecule and siRNAs can increase HDR efficiency to up to 93% and simultaneously prevent off-target editing.[78]
Delivery
Delivery of Cas9, sgRNA, and associated complexes into cells can occur via viral and non-viral systems. Electroporation of DNA, RNA, or ribonucleocomplexes is a common technique, though it can result in harmful effects on the target cells.[79] Chemical transfection techniques utilizing lipids and peptides have also been used to introduce sgRNAs in complex with Cas9 into cells.[80][81] Nanoparticle-based delivery has also been used for transfection.[82] Types of cells that are more difficult to transfect (e.g., stem cells, neurons, and hematopoietic cells) require more efficient delivery systems, such as those based on lentivirus (LVs), adenovirus (AdV), and adeno-associated virus (AAV).[83][84][85]
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.
Controlled genome editing
Further improvements and variants of the CRISPR-Cas9 system have focused on introducing more control into its use. Specifically, the research aimed at improving this system includes improving its specificity, its efficiency, and the granularity of its editing power. Techniques can further be divided and classified by the component of the system they modify. 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.
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. Minimizing off-target effects is thus maximizing the safety of the system. 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.[90][91] 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.[92][93] Iteratively truncated sgRNAs and highly stabilized gRNAs have been shown to also decrease off-target effects.[94][95] 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.[96][97]
Several variants of CRISPR-Cas9 allow gene activation or genome editing with an external trigger such as light or small molecules.[98][99][100] These include photoactivatable CRISPR systems developed by fusing light-responsive protein partners with an activator domain and a dCas9 for gene activation,[101][102] or by fusing similar light-responsive domains with two constructs of split-Cas9,[103][104] or by incorporating caged unnatural amino acids into Cas9,[105] or by modifying the guide RNAs with photocleavable complements for genome editing.[106]
Methods to control genome editing with small molecules include an allosteric Cas9, with no detectable background editing, that will activate binding and cleavage upon the addition of
CRISPR also utilizes single base-pair editing proteins to create specific edits at one or two bases in the target sequence. CRISPR/Cas9 was fused with specific enzymes that initially could only change C to T and G to A mutations and their reverse. This was accomplished eventually without requiring any DNA cleavage.[117][118][119] With the fusion of another enzyme, the base editing CRISPR-Cas9 system can also edit C to G and its reverse.[120]
CRISPR screening
The clustered regularly interspaced short palindrome repeats (CRISPR)/Cas9 system is a gene-editing technology that can induce double-strand breaks (DSBs) anywhere guide ribonucleic acids (gRNA) can bind with the protospacer adjacent motif (PAM) sequence.[121] Single-strand nicks can also be induced by Cas9 active-site mutants,[122] also known as Cas9 nickases.[123] By simply changing the sequence of gRNA, the Cas9-endonuclease can be delivered to a gene of interest and induce DSBs.[124] 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.[125][126] CRISPR screening helps scientist to create a systematic and high-throughput genetic perturbation within live model organisms. This genetic perturbation is necessary for fully understanding gene function and epigenetic regulation.[127] The advantage of pooled CRISPR libraries is that more genes can be targeted at once.
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.[121] There are two plasmid systems in CRISPR/Cas9 libraries. First, is all in one plasmid, where sgRNA and Cas9 are produced simultaneously in a transfected cell. Second, is a two-vector system: sgRNA and Cas9 plasmids are delivered separately.[127] 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.[125]
Once pooled library is prepared it is necessary to carry out a deep sequencing (NGS, next generation sequencing) of PCR-amplifed plasmid DNA in order to reveal abundance of sgRNAs. Cells of interest can be consequentially infected by the library and then selected according to the phenotype. There are 2 types of selection: negative and positive. By negative selection dead or slow growing cells are efficiently detected. It can identify survival-essential genes, which can be further serve as candidates for molecularly targeted drugs. On the other hand, positive selection gives a collection of growth-advantage acquired populations by random mutagenesis.[121] After selection genomic DNA is collected and sequenced by NGS. Depletion or enrichment of sgRNAs is detected and compared to the original sgRNA library, annotated with the target gene that sgRNA corresponds to. Statistical analysis then identify genes that are significantly likely to be relevant to the phenotype of interest.[125]
Library | ID | Species | PI | Genes targeted | gRNAs per gene | Total gRNAs |
---|---|---|---|---|---|---|
Bassik Mouse CRISPR Knockout Library | 1000000121–1000000130 | Mouse | Bassik | Varies (~23,000 in total) | ~10 | Varies |
Mouse Tumor Suppressor Gene CRISPR Knockout Library | 113584 EFS backbone
113585 TBG backbone |
Mouse | Chen | 56 | ~4 | 286 |
Brie mouse genome-wide library | 73632 (1 plasmid)
73633 (2 plasmid) |
Mouse | Doench and Root | 19,674 | 4 | 78,637 |
Bassik Human CRISPR Knockout Library | 101926–101934 | Human | Bassik | Varies (~20,500 in total) | ~10 | Varies |
Brunello human genome-wide library | 73179 (1 plasmid)
73178 (2 plasmid) |
Human | Doench and Root | 19,114 | 4 | 76,441 |
Mini-human AsCpf1-based Human Genome-wide Knockout Library | 130630 | Human | Draetta | 16,977 | 3–4 | 17,032 arrays |
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. It made CRISPR/Cas9 system even more interesting in gene editing. Inactive dCas9 protein modulate gene expression by targeting dCas9-repressors or activators toward promoter or transcriptional start sites of target genes. For repressing genes Cas9 can be fused to KRAB effector domain that makes complex with gRNA, whereas CRISPRa utilizes dCas9 fused to different transcriptional activation domains, which are further directed by gRNA to promoter regions to upregulate expression.[129][130][131]
Applications
Disease models
Cas9 genomic modification has allowed for the quick and efficient generation of transgenic models within the field of genetics. 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.[132] 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.
The CRISPR and Cas9 revolution in genomic modeling does not extend only to mammals. Traditional genomic models such as Drosophila melanogaster, one of the first model organisms, have seen further refinement in their resolution with the use of Cas9.[132] Cas9 uses cell-specific promoters allowing a controlled use of the Cas9. Cas9 is an accurate method of treating diseases due to the targeting of the Cas9 enzyme only affecting certain cell types. The cells undergoing the Cas9 therapy can also be removed and reintroduced to provide amplified effects of the therapy.[133]
CRISPR-Cas9 can be used to edit the DNA of organisms in vivo and to eliminate individual genes or even entire
Successful in vivo genome editing using CRISPR-Cas9 has been shown in numerous model organisms, including
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. the observed phenotypic change may not be due to modifying the target gene, but some other gene). Modifications to CRISPR have been made to minimize the possibility of off-target effects. Orthogonal CRISPR experiments are often recommended to confirm the results of a gene editing experiment.[147][148]
CRISPR simplifies the creation of genetically modified organisms for research which mimic disease or show what happens when a gene is knocked down or mutated. CRISPR may be used at the germline level to create organisms in which the targeted gene is changed everywhere (i.e. 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.[149][150][151]
CRISPR can be utilized to create human cellular models of disease.[152] For instance, when applied to human pluripotent stem cells, CRISPR has been used to introduce targeted mutations in genes relevant to polycystic kidney disease (PKD) and focal segmental glomerulosclerosis (FSGS).[153] These CRISPR-modified pluripotent stem cells were subsequently grown into human kidney organoids that exhibited disease-specific phenotypes. Kidney organoids from stem cells with PKD mutations formed large, translucent cyst structures from kidney tubules. The cysts were capable of reaching macroscopic dimensions, up to one centimeter in diameter.[154] Kidney organoids with mutations in a gene linked to FSGS developed junctional defects between podocytes, the filtering cells affected in that disease. This was traced to the inability of podocytes to form microvilli between adjacent cells.[155] Importantly, these disease phenotypes were absent in control organoids of identical genetic background, but lacking the CRISPR modifications.[153]
A similar approach was taken to model
Biomedicine
CRISPR-Cas technology has been proposed as a treatment for multiple human diseases, especially those with a genetic cause.[157] Its ability to modify specific DNA sequences makes it a tool with potential to fix disease-causing mutations. Early research in animal models suggest that therapies based on CRISPR technology have potential to treat a wide range of diseases,[158] including cancer,[159] progeria,[160] beta-thalassemia,[161][162][163] sickle cell disease,[163][164] hemophilia,[165] cystic fibrosis,[166] Duchenne's muscular dystrophy,[167] Huntington's disease,[168][169] transthyretin amyloidosis[43] and heart disease.[170] CRISPR has also been used to cure malaria in mosquitos, which could eliminate the vector and the disease in humans.[171] 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.[172]
In addition, clinical trials to cure beta thalassemia and sickle cell disease in human patients using CRISPR-Cas9 technology have shown promising results.[173][174]
Nevertheless, there remains a few limitations of the technology's use in
Ocular Diseases
Leber Congenital Amaurosis:
The CRISPR treatment for LCA10 (the most common variant of Leber Congenital Amaurosis which is the leading cause of inherited childhood blindness) modifies the patient's defective photoreceptor gene.
In March 2020, the first patient volunteer in this US-based study, sponsored by Editas Medicine, was given a low-dose of the treatment to test for safety.
In June 2021, enrollment began for a high-dose adult and pediatric cohort of 4 patient volunteers each. Dosing of the new cohorts is expected to be completed by July 2022.[176] In November 2022, Editas reported that 20% of the patients treated had significant improvements, but also announced that the resulting target population was too small to support continued independent development.[177]
Herpes Simplex Ophthalmicus:
Herpes Simplex Ophthalmicus(HSK) is caused by a viral infection of Herpes Simplex Virus type 1(HSV-1) or type 2(HSV-2) with a primary and secondary level of infection that lead to affecting all structures of the eye.[178] While early detection and treatment is possible with antiviral agents, once a patient is infected it is nearly impossible to completely eradicate from the body, and severe cases can lead to serious visual impairment/blindness.[178]
Cancer
CRISPR has also found many applications in developing cell-based immunotherapies.[179] The first clinical trial involving CRISPR started in 2016. It involved taking immune cells from people with lung cancer, using CRISPR to edit out the gene expressed PD-1, then administering the altered cells back to the same person. 20 other trials were under way or nearly ready, mostly in China, as of 2017[update].[159]
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.[180]
In November 2020, in mouse animal models, CRISPR was used effectively to treat
In October 2021, CRISPR Therapeutics announced results from their ongoing US-based Phase 1 trial for an allogeneic T cell therapy. 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.[176]
In December 2022, a 13-year British girl that had been diagnosed with incurable
Diabetes
Type 1 Diabetes is an endocrine disorder which results from a lack of pancreatic beta cells to produce insulin, a vital compound in transporting blood sugar to cells for producing energy. Researchers have been trying to transplant healthy beta cells. CRISPR is used to edit the cells in order to reduce the chance the patient's body will reject the transplant.
On November 17, 2021 CRISPR therapeutics and ViaCyte announced that the Canadian medical agency had approved their request for a clinical trial for VCTX210, a CRISPR-edited stem cell therapy designed to treat type 1 diabetes. This was significant because it was the first ever gene-edited therapy for diabetes that approached clinics. The same companies also developed a novel treatment for type 1 diabetes to produce insulin via a small medical implant that uses millions of pancreatic cells derived from CRISPR gene-edited stem cells.[184]
In February 2022, a phase 1 trial was conducted in which one patient volunteer received treatment.[176][185]
HIV/AIDS
Human immunodeficiency virus or HIV, is a virus that attacks the body's immune system. 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. CRISPR can be used to selectively remove the virus from the genome by designing guide RNA to target the retroactive HIV genome. 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.[176]
Initial results in the treatment and cure of HIV have been rather successful, in 2021 9 out of 23 humanized mice treated with a combination of anti-retrovirals and CRISPR/Cas-9 which lead to the virus becoming undetectable, even after the usual rebound period. None of the two treatments alone had such an effect.[186] Clinical trials in humans of a CRISPR–Cas9 based therapy, EBT-101 are to start in 2022.[187][188] In October 2023 an early-stage study on 3 people of EBT-101 reported that the treatment appeared to be safe with no major side effects but no data on its effectiveness was disclosed.[189] In 2024 another CRISPR therapy from researchers of the university of Amsterdam reported the elimination of HIV on cell cultures. In March 2024 researchers from the university of Amsterdam reported the elimination of HIV on cell cultures using CRISPR.[190][191]
Infection
CRISPR-Cas-based "RNA-guided nucleases" can be used to target
Therapies based on CRISPR–Cas3 gene editing technology delivered by engineered bacteriophages could be used to destroy targeted DNA in pathogens.[195] Cas3 is more destructive than the better known Cas9.[196][197]
Research suggests that CRISPR is an effective way to limit replication of multiple
CRISPR may revive the concept of transplanting animal organs into people. Retroviruses present in animal genomes could harm transplant recipients. In 2015, a team eliminated 62 copies of a particular retroviral DNA sequence from the pig genome in a kidney epithelial cell.[199] Researchers recently demonstrated the ability to birth live pig specimens after removing these retroviruses from their genome using CRISPR for the first time.[200]
Neurological Diseases
CRISPR is unique to the development of solving neurological diseases for several reasons. For example, CRISPR allows researches to quickly generate animal and human cell models. This allows them to study how genes function in a nervous system. By introducing mutations that pertain to various diseases within these cells, researches can study the effects of the changes on nervous system development, function, and behavior.[201] They can uncover the molecular mechanisms that contribute to these disorders, which is essential for developing effective treatments. This is particularly useful in modeling and treating complex neurological disorders such as Alzheimer's, Parkinson's, and epilepsy among other.
Alzheimer's Disease (AD) is a neurodegenerative disease categorized by neuron loss and an accumulation of intracellular neurofibrillary tangles and extracellular amyloid plaques in the brain.[202] Three known pathogenic genes that cause early onset AD in humans has been identified, specifically amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2).[202] Over 300 mutations have ben detected in these genes, resulting in an increase in total β-amyloid (Aβ), Aβ42/40 ratio, and/or Aβ polymerization.
In the case of Duchenne muscular dystrophy, the mutation responsible for the disease occurs in the dystrophin gene.[203] CRISPR has been used to correct for this. Similarly, for Dravet syndrome, which is an epilepsy disorder, CRISPR has been used to correct the SCN1A gene mutation.[204] Despite the progress that has been made, there are still challenges around using CRISPR. Due to the fact the brain is composed of a blood-brain barrier, it is difficult to transfer CRISPR components across this. However, recent advancements in nanoparticle delivery systems and viral vectors have shown promise in overcoming this hurdle. Looking in the future, the use of CRISPR in neuroscience is expected to increase as technology evolves.
Genetic anthropology
CRISPR-Cas9 can be used in investigating and identifying the genetic differences of humans to other apes, especially
By technique
Knockdown/activation
Using "dead" versions of Cas9 (dCas9) eliminates CRISPR's DNA-cutting ability, while preserving its ability to target desirable sequences. Multiple groups added various regulatory factors to dCas9s, enabling them to turn almost any gene on or off or adjust its level of activity.[199] Like RNAi, CRISPR interference (CRISPRi) turns off genes in a reversible fashion by targeting, but not cutting a site. The targeted site is methylated, epigenetically modifying the gene. This modification inhibits transcription. These precisely placed modifications may then be used to regulate the effects on gene expressions and DNA dynamics after the inhibition of certain genome sequences within DNA. Within the past few years, epigenetic marks in different human cells have been closely researched and certain patterns within the marks have been found to correlate with everything ranging from tumor growth to brain activity.[10] Conversely, CRISPR-mediated activation (CRISPRa) promotes gene transcription.[213] Cas9 is an effective way of targeting and silencing specific genes at the DNA level.[214] In bacteria, the presence of Cas9 alone is enough to block transcription. For mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA sequences called promoters that immediately precede the target gene.[215]
Cas9 was used to carry synthetic transcription factors that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different locations on the gene's promoter.[215]
RNA editing
In 2016, researchers demonstrated that CRISPR from an ordinary mouth bacterium could be used to edit RNA. The researchers searched databases containing hundreds of millions of genetic sequences for those that resembled CRISPR genes. They considered the fusobacterium Leptotrichia shahii. It had a group of genes that resembled CRISPR genes, but with important differences. When the researchers equipped other bacteria with these genes, which they called C2c2, they found that the organisms gained a novel defense.[216] C2c2 has later been renamed to Cas13a to fit the standard nomenclature for Cas genes.[217]
Many viruses encode their genetic information in RNA rather than DNA that they repurpose to make new viruses. HIV and poliovirus are such viruses. Bacteria with Cas13 make molecules that can dismember RNA, destroying the virus. Tailoring these genes opened any RNA molecule to editing.[216]
CRISPR-Cas systems can also be employed for editing of micro-RNA and long-noncoding RNA genes in plants.[218]
Therapeutic applications
Directing edits to correct mutated sequences was first proposed and demonstrated in 1995.[219] This initial work used synthetic RNA antisense oligonucleotides complementary to a pre-mature stop codon mutation in a dystrophin sequence to activate A-to-I editing of the stop codon to a read through codon in a model xenopus cell system.[219] While this also led to nearby inadvertent A-to-I transitions, A to I (read as G) transitions can correct all three stop codons, but cannot create a stop codon. Therefore, the changes led >25% correction of the targeted stop codon with read through to a downstream luciferase reporter sequence. Follow on work by Rosenthal achieved editing of mutated mRNA sequence in mammalian cell culture by directing an oligonucleotide linked to a cytidine deaminase to correct a mutated cystic fibrosis sequence.[220] More recently, CRISPR-Cas13 fused to deaminases has been employed to direct mRNA editing.[221]
In 2022, therapeutic RNA editing for Cas7-11 was reported.[222][223] It enables sufficiently targeted cuts and an early version of it was used for in vitro editing in 2021.[224]Comparison to DNA editing
Gene drive
Gene drives may provide a powerful tool to restore balance of ecosystems by eliminating invasive species. Concerns regarding efficacy, unintended consequences in the target species as well as non-target species have been raised particularly in the potential for accidental release from laboratories into the wild. Scientists have proposed several safeguards for ensuring the containment of experimental gene drives including molecular, reproductive, and ecological.[226] Many recommend that immunization and reversal drives be developed in tandem with gene drives in order to overwrite their effects if necessary.[227] There remains consensus that long-term effects must be studied more thoroughly particularly in the potential for ecological disruption that cannot be corrected with reversal drives.[228]
In vitro genetic depletion
Unenriched sequencing libraries often have abundant undesired sequences. Cas9 can specifically deplete the undesired sequences with double strand breakage with up to 99% efficiency and without significant off-target effects as seen with restriction enzymes. Treatment with Cas9 can deplete abundant rRNA while increasing pathogen sensitivity in RNA-seq libraries.[229]
Epigenome editing
Epigenome editing or epigenome engineering is a type of genetic engineering in which the epigenome is modified at specific sites using engineered molecules targeted to those sites (as opposed to whole-genome modifications). Whereas gene editing involves changing the actual DNA sequence itself, epigenetic editing involves modifying and presenting DNA sequences to proteins and other DNA binding factors that influence DNA function. By "editing” epigenomic features in this manner, researchers can determine the exact biological role of an epigenetic modification at the site in question.
The engineered proteins used for epigenome editing are composed of a DNA binding domain that target specific sequences and an effector domain that modifies epigenomic features. Currently, three major groups of DNA binding proteins have been predominantly used for epigenome editing:Applications
Targeted regulation of disease-related genes may enable novel therapies for many diseases, especially in cases where adequate gene therapies are not yet developed or are inappropriate.[230] While transgenerational and population level consequences are not fully understood, it may become a major tool for applied functional genomics and personalized medicine.[231] As with RNA editing, it does not involve genetic changes and their accompanying risks.[230] One example of a potential functional use of epigenome editing was described in 2021: repressing Nav1.7 gene expression via CRISPR-dCas9 which showed therapeutic potential in three mouse models of chronic pain.[232][233]
In 2022, research assessed its usefulness in reducing tau protein levels, regulating a protein involved in Huntington's disease, targeting an inherited form of obesity, and Dravet syndrome.[234]CRISPR-directed integrases
Combination of CRISPR-Cas9 with integrases enabled a technique for large edits without problematic double-stranded breaks, as demonstrated with PASTE in 2022. The researchers reported it could be used to deliver genes as long as 36,000 DNA base pairs to several types of human cells and thereby potentially for treating diseases caused by a large number of mutations.[235][236]
Prime editing
Prime editing[237] (or base editing) is a CRISPR refinement to accurately insert or delete sections of DNA. The CRISPR edits are not always perfect and the cuts can end up in the wrong place. Both issues are a problem for using the technology in medicine.[238] Prime editing does not cut the double-stranded DNA but instead uses the CRISPR targeting apparatus to shuttle an additional enzyme to a desired sequence, where it converts a single nucleotide into another.[239] The new guide, called a pegRNA, contains an RNA template for a new DNA sequence to be added to the genome at the target location. That requires a second protein, attached to Cas9: a reverse transcriptase enzyme, which can make a new DNA strand from the RNA template and insert it at the nicked site.[240] Those three independent pairing events each provide an opportunity to prevent off-target sequences, which significantly increases targeting flexibility and editing precision.[239] Prime editing was developed by researchers at the Broad Institute of MIT and Harvard in Massachusetts.[241] More work is needed to optimize the methods.[241][240]
Society and culture
Human germline modification
As of March 2015, multiple groups had announced ongoing research with the intention of laying the foundations for applying CRISPR to human embryos for human germline engineering, including labs in the US, China, and the UK, as well as US biotechnology company OvaScience.[242] Scientists, including a CRISPR co-discoverer, urged a worldwide moratorium on applying CRISPR to the human germline, especially for clinical use. They said "scientists should avoid even attempting, in lax jurisdictions, germline genome modification for clinical application in humans" until the full implications "are discussed among scientific and governmental organizations".[243][244] These scientists support further low-level research on CRISPR and do not see CRISPR as developed enough for any clinical use in making heritable changes to humans.[245]
In April 2015, Chinese scientists reported results of an attempt to alter the DNA of non-viable
In December 2015, an International Summit on Human Gene Editing took place in Washington under the guidance of
In February 2017, the
In November 2018,
Designer Babies
The advent of CRISPR-Cas9 gene editing technology has led to the possibility of creating "designer babies." This technology has the possibility of eliminating certain genetic diseases, or improving health by enhancing certain genetic traits.
Policy barriers to genetic engineering
Policy regulations for the CRISPR-Cas9 system vary around the globe. In February 2016, British scientists were given permission by regulators to genetically modify
The US has an elaborate, interdepartmental regulatory system to evaluate new genetically modified foods and crops. For example, the
In 2016, the USDA sponsored a committee to consider future regulatory policy for upcoming genetic modification techniques. With the help of the US National Academies of Sciences, Engineering, and Medicine, special interests groups met on April 15 to contemplate the possible advancements in genetic engineering within the next five years and any new regulations that might be needed as a result.[258] In 2017, the Food and Drug Administration proposed a rule that would classify genetic engineering modifications to animals as "animal drugs", subjecting them to strict regulation if offered for sale and reducing the ability for individuals and small businesses to make them profitable.[259][260]
In China, where social conditions sharply contrast with those of the West, genetic diseases carry a heavy stigma.[261] This leaves China with fewer policy barriers to the use of this technology.[262][263]
Recognition
In 2012 and 2013, CRISPR was a runner-up in
See also
- CRISPR/Cas Tools
- The CRISPR Journal
- Eugenics
- DRACO
- Zinc finger
- Gene knockout
- Genetics
- Glossary of genetics
- Human Nature (2019 documentary film)
- LEAPER gene editing
- Make People Better (2022 documentary)
- RNAi
- SiRNA
- Surveyor nuclease assay
- Synthetic biology
References
- ^ S2CID 49269023.
- ^ "The Nobel Prize in Chemistry 2020". The Nobel Prize. Retrieved 2020-12-10.
- S2CID 225116732.
- ^ Cohen J (2018-06-04). "With prestigious prize, an overshadowed CRISPR researcher wins the spotlight". Science | AAAS. Retrieved 2020-05-02.
- ^ a b Owens R (8 October 2020). "Nobel prize: who gets left out?". The Conversation. Retrieved 13 December 2021.
- ^ "Lithuanian scientists not awarded Nobel prize despite discovering same technology". LRT.LT. 8 October 2020.
- ^ Šikšnys V (2018-06-16). "Imam genų žirkles, iškerpam klaidą, ligos nelieka". Laisvės TV / Freedom TV. 12:22 minutes in. LaisvėsTV. <...>Tai mes tą savo straipsnį išsiuntėm į redakciją pirmieji, bet laimės ten daug nebuvo. Viena redakcija pasakė, kad mes net recenzentam nesiųsim. Nusiuntėm į kitą redakciją – tai jis (straipsnis) pragulėjo kažkur ant redaktoriaus stalo labai ilgai. Na ir taip galų gale išsiuntėm į trečią žurnalą ir trečias žurnalas po kelių mėnesių jį išspausdino. Bet, aišku, Berklio universiteto mokslininkams sekėsi geriau – jie išsiuntė straipsnį į žurnalą Science – jį priėmė ir išspausdino per 2 savaites. Nors iš tikro jie tą straispnį išsiuntė pora mėnesių vėliau nei mes. Retrieved 2018-06-30.
<...> Well, we were who had sent the article first, but had not much of luck.
- PMID 25193712.
- PMID 30082871.
- ^ PMID 26961639.
- ^ Travis J (17 December 2015). "Breakthrough of the Year: CRISPR makes the cut". Science Magazine. American Association for the Advancement of Science.
- PMID 26040877.
- ^ a b "Casgevy: UK approves gene-editing drug for sickle cell". BBC News. 16 November 2023. Retrieved 16 November 2023.
- ^ a b "MHRA authorises world-first gene therapy that aims to cure sickle-cell disease and transfusion-dependent β-thalassemia". Gov.uk. 16 November 2023. Retrieved 16 November 2023.
- ^ "FDA Approves First Gene Therapies to Treat Patients with Sickle Cell Disease". Food and Drug Administration. 11 December 2023. Retrieved 11 December 2023.
- ^ Young S (11 February 2014). "CRISPR and Other Genome Editing Tools Boost Medical Research and Gene Therapy's Reach". MIT Technology Review. Retrieved 2014-04-13.
- ^ PMID 26656253.
- S2CID 21543486.
- PMID 25654603.
- ^ a b c d "CRISPR Timeline". Broad Institute. 2015-09-25. Retrieved 2023-12-08.
- ^ "CRISPR Madness". GEN. 2013-11-08.
- .
- Technology Review. Retrieved 25 February 2015.
- ^ Fye S. "Genetic Rough Draft: Editas and CRISPR". The Atlas Business Journal. Retrieved 19 January 2016.
- ^ "CRISPR-Cas systems and methods for altering expression of gene products". Google Patents.
- S2CID 247453528.
- S2CID 205265912.
- ^ Pollack A (15 February 2017). "Harvard and M.I.T. Scientists Win Gene-Editing Patent Fight". The New York Times.
- ^ Akst J (February 15, 2017). "Broad Wins CRISPR Patent Interference Case". The Scientist Magazine.
- ^ Noonan KE (February 16, 2017). "PTAB Decides CRISPR Interference in Favor of Broad Institute – Their Reasoning". Patent Docs.
- ^ Potenza A (April 13, 2017). "UC Berkeley challenges decision that CRISPR patents belong to Broad Institute". The Verge. Retrieved 22 September 2017.
- ^ Buhr S (July 26, 2017). "The CRISPR patent battle is back on as UC Berkeley files an appeal". TechCrunch. Retrieved 22 September 2017.
- ^ Westman N (March 1, 2022). "UC Berkeley loses CRISPR patent case". The Verge. Retrieved March 6, 2022.
- ^ a b Philippidis A (August 7, 2017). "MilliporeSigma to Be Granted European Patent for CRISPR Technology". Genetic Engineering & Biotechology News. Retrieved 22 September 2017.
- ^ Akst J (March 24, 2017). "UC Berkeley Receives CRISPR Patent in Europe". The Scientist. Retrieved 22 September 2017.
- .
- ^ "Top EU court: GMO rules cover plant gene editing technique". Retuers. 25 July 2018.
- ^ AFP (7 February 2020). "US Trial Shows 3 Cancer Patients Had Their Genomes Altered Safely by CRISPR". ScienceAlert. Retrieved 2020-02-09.
- ^ Chamary JV. "These Scientists Deserved A Nobel Prize, But Didn't Discover Crispr". Forbes. Retrieved 2020-07-10.
- ^ Fischman J. "Nobel Prize in Chemistry Goes to Discovery of 'Genetic Scissors' Called CRISPR/Cas9". Scientific American. Retrieved 2021-03-24.
- ^ "Two women share chemistry Nobel in historic win for 'genetic scissors'". BBC News. 2020-10-07. Retrieved 2020-12-06.
- ^ Kaiser J (26 June 2021). "CRISPR injected into the blood treats a genetic disease for first time". Science | AAAS. Retrieved 11 July 2021.
- ^ S2CID 235722446.
- PMID 26500584.
- ^ "Tomato In Japan Is First CRISPR-Edited Food In The World To Go On Sale". IFLScience. Retrieved 18 October 2021.
- PMID 31240102.
- S2CID 245593283.
- ^ "Startup hopes genome-edited pufferfish will be a hit in 2022". The Japan Times. 5 January 2022. Archived from the original on 17 January 2022. Retrieved 17 January 2022.
- ^ "Gene-edited sea bream set for sale in Japan". thefishsite.com.
- hdl:10419/249208.
- ^ "Are Consumers Willing to Buy CRISPR Tomatoes?". Crop Biotech Update. Retrieved 2022-02-21.
- ^ Whitford E (2021-05-28). "UC Berkeley Will Auction NFTs for 2 Nobel Prize Patents". Inside Higher Ed. Retrieved 2023-02-21.
- S2CID 250238540.
- ^ Chang K (2021-05-27). "You Can Buy a Piece of a Nobel Prize-Winning Discovery". New York Times. Retrieved 2023-02-21.
- S2CID 234830426.
- S2CID 235481285.
- ^ Office of the Commissioner (2023-12-08). "FDA Approves First Gene Therapies to Treat Patients with Sickle Cell Disease". FDA. Retrieved 2023-12-14.
- ^ PMID 24157548.
- ^ Ly J (2013). Discovering Genes Responsible for Kidney Diseases (Ph.D.). University of Toronto. Retrieved 26 December 2016.
- PMID 27276584.
- PMID 26745836.
- ^ "Researchers establish new viable CRISPR-Cas12b system for plant genome engineering". phys.org. Retrieved 6 April 2020.
- S2CID 212643374.
- PMID 29070703.
- ^ "CRISPR-Cas3 innovation holds promise for disease cures, advancing science". Cornell Chronicle. Retrieved 24 October 2021.
- PMID 30975459.
- PMID 32883277.
- ^ "Researchers develop an engineered 'mini' CRISPR genome editing system". phys.org. Retrieved 18 October 2021.
- S2CID 237417317.
- PMID 35236982.
- ^ "Protein tweak makes CRISPR gene editing 4,000 times less error-prone". New Atlas. 2022-03-04. Retrieved 2022-03-07.
- S2CID 249103058.
- Lay summary: Williams S. "Neuroscientists expand CRISPR toolkit with new, compact Cas7-11 enzyme". Massachusetts Institute of Technology. Retrieved 22 June 2022.
- ^ "'Softer' form of CRISPR may edit genes more accurately". New Scientist. Retrieved 21 August 2022.
- PMID 35776792.
- S2CID 17960960.
- PMID 26053390.
- PMID 24584096.
- ^ PMID 37474806.
- PMID 29801422.
- PMID 29704747.
- PMID 31670340.
- ^ PMID 32486234.
- PMID 28723575.
- S2CID 37653318.
- ^ Waxmonsky N (24 September 2015). "CRISPR 101: Mammalian Expression Systems and Delivery Methods". Retrieved 11 June 2018.
- PMID 35402072.
- PMID 29765029.
- PMID 27848933.
- S2CID 17028472.
- PMID 35301321.
- PMID 26735016.
- PMID 26628643.
- PMID 29377001.
- S2CID 246281892.
- PMID 24463574.
- PMID 35468907.
- PMID 24253446.
- ^ PMID 27136077.
- PMID 26857072.
- PMID 26996256.
- PMID 25664691.
- PMID 25619936.
- PMID 25713377.
- S2CID 205281536.
- PMID 25905628.
- PMID 27554600.
- PMID 25848930.
- S2CID 33891039.
- PMID 26082496.
- PMID 25643054.
- PMID 24931489.
- PMID 25690852.
- PMID 25658371.
- S2CID 256273893.
- PMID 25798939.
- ^ PMID 29610531.
- S2CID 5122081.
- PMID 27096365.
- PMID 29160308.
- PMID 33654077.
- ^ S2CID 3308058.
- PMID 22949671.
- PMID 28522803.
- PMID 31971562.
- ^ PMID 26442115.
- S2CID 140275157.
- ^ PMID 28333914.
- ^ "Addgene: Pooled Libraries". www.addgene.org. Retrieved 2020-01-31.
- S2CID 5055878.
- PMID 23979020.
- PMID 25307932.
- ^ PMID 26432018.
- )
- PMID 29178945.
- "CRISPR Used to Eliminate Targeted Chromosomes in New Study". Genome Web. Nov 27, 2017.
- S2CID 51920661.
- PMID 23460208.
- PMID 29259528.
- PMID 32826220.
- PMID 29331410.
- ^ PMID 26762955.
- PMID 28879855.
- PMID 30076892.
- PMID 25271304.
- PMID 29796309.
- PMID 28677007.
- PMID 29258297.
- S2CID 4846191.
- S2CID 52078796.
- PMID 25914022.
- PMID 25819765.
- PMID 25868999.
- ^ "What Is CRISPR? How Does It Work? Is It Gene Editing?". LiveScience.Tech. 2018-04-30. Archived from the original on 2020-02-06. Retrieved 2020-02-06.
- ^ PMID 26493500.
- PMID 28967916.
- PMID 28905451.
- PMID 24213244.
- PMID 30258895.
- ^ "Seven Diseases That CRISPR Technology Could Cure". Labiotech.eu. 2018-06-25. Retrieved 2018-08-22.
- ^ a b "CRISPR/Cas9 and Cancer". Immuno-Oncology News. 2018-04-27. Retrieved 2019-02-18.
- ^ Crossley M (February 2021). "New CRISPR technology could revolutionise gene therapy, offering new hope to people with genetic diseases". The Conversation. Retrieved 2021-02-03.
- PMID 33737751.
- PMID 25096406.
- ^ S2CID 227521558.
- PMID 27820943.
- ^ "CRISPR 'One Shot' Cell Therapy for Hemophilia Developed". GEN. 2018-05-02. Retrieved 2018-08-22.
- PMID 29731717.
- PMID 28195574.
- PMID 29844549.
- PMID 29535594.
- PMID 29517035.
- PMID 31289403.
- PMID 25940550.
- ^ "A Year In, 1st Patient To Get Gene Editing For Sickle Cell Disease Is Thriving". NPR.org. Retrieved 2021-02-03.
- ^ "CRISPR technology to cure sickle cell disease". ScienceDaily. Retrieved 2021-02-03.
- PMID 32850447.
- ^ a b c d "CRISPR Clinical Trials: A 2022 Update". Innovative Genomics Institute (IGI). Retrieved 2022-05-02.
- ^ "Editas Hits Pause on LCA10 Program in Search of Partner".
- ^ PMID 32644620. Retrieved 2024-01-12.
- S2CID 76663873.
- S2CID 89466280.
- PMID 33208369.
- ^ "Base editing: Revolutionary therapy clears girl's incurable cancer". BBC News. 11 December 2022. Retrieved 11 December 2022.
- ^ "'Designer cells' reverse one-year-old's cancer". BBC News. 2015-11-05. Retrieved 2022-12-11.
- ^ May 12 (2022-05-12). "B.C. researchers launching clinical trial for first genetically engineered stem cell-based therapy for type 1 diabetes". UBC News. Retrieved 2023-12-08.
{{cite web}}
: CS1 maint: numeric names: authors list (link) - ^ "CRISPR Therapeutics and ViaCyte, Inc. Announce First Patient Dosed in…". CRISPR. Retrieved 2022-05-02.
- ^ National Institute on Drug Abuse (2020-02-14). "Antiretroviral Therapy Combined With CRISPR Gene Editing Can Eliminate HIV Infection in Mice". National Institute on Drug Abuse. Retrieved 2022-04-18.
- ^ "First Clinical Trial of CRISPR-Based HIV Therapy Founded on Breakthrough Research at Lewis Katz School of Medicine | Lewis Katz School of Medicine at Temple University". medicine.temple.edu. Retrieved 2022-04-18.
- ^ Excision BioTherapeutics (2022-03-24). "A Phase 1/2a, Sequential Cohort, Single Ascending Dose Study of the Safety, Tolerability, Biodistribution, and Pharmacodynamics of EBT 101 in Aviremic HIV-1 Infected Adults on Stable Antiretroviral Therapy".
- ^ "Three people were gene-edited in an effort to cure their HIV. The result is unknown". MIT Technology Review. Retrieved 2024-03-20.
- ^ "HIV in cell culture can be completely eliminated using CRISPR-Cas gene editing technology, increasing hopes of cure". EurekAlert!. Retrieved 2024-03-20.
- ^ "Scientists say they can cut HIV out of cells". 2024-03-20. Retrieved 2024-03-20.
- PMID 24473129.
- PMID 25240928.
- ^ S2CID 22716314.
- PMID 29300040.
- ^ Taylor P (Jan 3, 2019). "J&J takes stake in Locus' CRISPR-based 'Pac-Man' antimicrobials". Fierce Biotech. Retrieved 27 February 2019.
- PMID 28661508.
- PMID 27362483.
- "Using CRISPR to combat viral infections: a new way to treat herpes?". PLOS Media. August 4, 2016 – via YouTube.
- ^ a b c Science News Staff (December 17, 2015). "And Science's Breakthrough of the Year is ..." news.sciencemag.org. Retrieved 2015-12-21.
- ^ Mullin E. "Using CRISPR on pigs could make their organs safer for human transplant". MIT Technology Review. Retrieved 2017-09-09.
- ^ Feijo S. "How Brown neuroscientists are using CRISPR to accelerate brain research — and more". Brown University. Retrieved 2023-12-08.
- ^ PMID 35955847.
- PMID 35163754.
- S2CID 218762990.
- S2CID 252161562.
- PMID 35905187.
- ^ "Scientists discover how humans develop larger brains than other apes". phys.org. Archived from the original on 19 April 2021. Retrieved 19 April 2021.
- .
- ^ Sawal I. "Mini brains genetically altered with CRISPR to be Neanderthal-like". New Scientist. Archived from the original on 10 March 2021. Retrieved 7 March 2021.
- PMID 33574182.
- S2CID 238990790.
- S2CID 238990560.
- PMID 26670017.
- PMID 24336571.
- ^ PMID 23970676.
- ^ ISSN 0362-4331. Retrieved 2016-06-10.
- PMID 31147612.
- PMID 26635829.
- ^ PMID 7545300.
- PMID 24108353.
- PMID 29070703.
- ^ Williams S. "Neuroscientists expand CRISPR toolkit with new, compact Cas7-11 enzyme". Massachusetts Institute of Technology. Retrieved 22 June 2022.
- S2CID 249103058.
- S2CID 237432753.
- ^ Cross R (25 March 2019). "Watch out, CRISPR. The RNA editing race is on". Chemical & Engineering News. 97 (12). Retrieved 30 September 2020.
- PMID 26229113.
- PMID 26450575.
- PMID 25035410.
- PMID 26944702.
- ^ PMID 26732754. Retrieved 30 April 2021.
- PMID 26388942.
- ^ "Unique CRISPR gene therapy offers opioid-free chronic pain treatment". New Atlas. 11 March 2021. Retrieved 18 April 2021.
- S2CID 232170826.
- ^ KAISER, JOCELYN (1 June 2022). "Better than CRISPR? Another way to fix gene problems may be safer and more versatile". www.science.org. Retrieved 2022-08-21.
- ^ McDonnell S. "New CRISPR-based tool inserts large DNA sequences at desired sites in cells". Massachusetts Institute of Technology via phys.org. Retrieved 18 December 2022.
- S2CID 253879386.
- PMID 31634902.
- ^ Thulin L (21 October 2019). "A New Gene Editing Tool Could Make CRISPR More Precise". The Smithsonian Magazine.
- ^ a b Cohen J (21 October 2019). "New 'prime' genome editor could surpass CRISPR". Science Magazine.
- ^ a b Yasinski E (21 October 2019). "New "Prime Editing" Method Makes Only Single-Stranded DNA Cuts". The Scientist.
- ^ a b Gallagher J (21 October 2019). "Prime editing: DNA tool could correct 89% of genetic defects". BBC News.
- ^ Regalado A (March 5, 2015). "Engineering the Perfect Baby". MIT Technology Review. Archived from the original on March 13, 2015.
- PMID 25791083.
- PMID 25810189.
- ^ Wade N (19 March 2015). "Scientists Seek Ban on Method of Editing the Human Genome". The New York Times. Retrieved 20 March 2015.
The biologists writing in Science support continuing laboratory research with the technique, and few if any scientists believe it is ready for clinical use.
- PMID 25894090.
- ^ Kolata G (23 April 2015). "Chinese Scientists Edit Genes of Human Embryos, Raising Concerns". The New York Times. Retrieved 24 April 2015.
- ^ S2CID 87604469.
- ^ Regalado A (2016-05-08). "Chinese Researchers Experiment with Making HIV-Proof Embryos". MIT Technology Review. Retrieved 2016-06-10.
- ^ "International Summit on Gene Editing". National Academies of Sciences, Engineering, and Medicine. 3 December 2015. Retrieved 3 December 2015.
- PMID 31021208.
- ^ Begley S (28 November 2018). "Amid uproar, Chinese scientist defends creating gene-edited babies". STAT.
- ^ Sample I (13 March 2019). "Scientists call for global moratorium on gene editing of embryos". Theguardian.com. Retrieved 14 March 2019.
- PMID 26842037.
- S2CID 3210837.
- USDA. "Re: Request to confirm"(PDF).
- PMID 27111611.
- PMID 27075074.
- ^ Brown KV (1 February 2017). "The FDA Is Cracking Down On Rogue Genetic Engineers". Gizmodo. Retrieved 5 February 2017.
- ^ "Guidance for Industry #187 / Regulation of Intentionally Altered Genomic DNA in Animals" (PDF). Food and Drug Administration. 2020-02-11.
- PMID 28816265.
- S2CID 38509820.
- ISSN 0099-9660. Retrieved 2018-01-23.
- ^ Talbot D (2016). "Precise Gene Editing in Plants/ 10 Breakthrough Technologies 2016". MIT Technology review. Massachusetts Institute of Technology. Archived from the original on November 5, 2017. Retrieved 18 March 2016.
- ^ Larson C, Schaffer A (2014). "Genome Editing/ 10 Breakthrough Technologies 2014". Massachusetts Institute of Technology. Archived from the original on December 5, 2016. Retrieved 18 March 2016.
- ^ "Tang Prize Laureates". www.tang-prize.org. Retrieved 2018-08-05.
- ^ "Press release: The Nobel Prize in Chemistry 2020". Nobel Foundation. Retrieved 7 October 2020.