CRISPR gene editing

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CRISPR-Cas9 GRNA-Cas9.png
CRISPR-Cas9

CRISPR gene editing 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 (in organisms that are alive). [1]

Contents

Synopsis

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. [2] 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. [2] 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 in the repair of the double-stranded break by means of non-homologous end joining (NHEJ). NHEJ 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. Because of this, the precision of genome editing is a great concern. Genomic editing leads to irreversible changes to the genome.

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. With the discovery of CRISPR and specifically the Cas9 nuclease molecule, efficient and highly selective editing is now a reality. 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. [3] 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. Newly engineered variants of the Cas9 nuclease have been developed that significantly reduce off-target activity. [4]

CRISPR-Cas9 genome editing techniques have many potential applications, including in medicine and agriculture. The use of the CRISPR-Cas9-gRNA complex for genome editing [5] was the AAAS's choice for Breakthrough of the Year in 2015. [6] Many bioethical concerns have been raised about the prospect of using CRISPR for germline editing, especially in human embryos. [7]

History

Predecessors

In the early 2000s, researchers developed zinc finger nucleases (ZFNs), synthetic proteins whose DNA-binding domains enable them to create double-stranded breaks in DNA at specific points. In 2010, synthetic nucleases called transcription activator-like effector nucleases (TALENs) provided an easier way to target a double-stranded break to a specific location on the DNA strand. 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. 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. creating oligonucleotide primers). [8]

Whereas methods such as RNA interference (RNAi) do not fully suppress gene function, CRISPR, ZFNs, and TALENs provide full irreversible gene knockout. [9] CRISPR can also target several DNA sites simultaneously simply by introducing different gRNAs. In addition, the costs of employing CRISPR are relatively low. [9] [10] [11]

Patents and commercialization

As of November 2013, 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. [12] By 2015, Thermo Fisher Scientific had licensed intellectual property from ToolGen to develop CRISPR reagent kits. [13]

As of December 2014, patent rights to CRISPR were contested. Several companies formed to develop related drugs and research tools. [14] As companies ramp up financing, doubts as to whether CRISPR can be quickly monetized were raised. [15] In February 2017 the US Patent Office ruled on a patent interference case brought by University of California with respect to patents issued to the Broad Institute, and found that the Broad patents, with claims covering the application of CRISPR-Cas9 in eukaryotic cells, were distinct from the inventions claimed by University of California. [16] [17] [18] Shortly after, University of California filed an appeal of this ruling. [19] [20]

Recent events

In March 2017, the European Patent Office (EPO) announced its intention to allow broad claims for editing all kinds of cells to Max-Planck Institute in Berlin, University of California, and University of Vienna, [21] [22] and in August 2017, the EPO announced its intention to allow CRISPR claims in a patent application that MilliporeSigma had filed. [21] As of August 2017 the patent situation in Europe was complex, with MilliporeSigma, ToolGen, Vilnius University, and Harvard contending for claims, along with University of California and Broad. [23]

In July 2018, the ECJ ruled that gene editing for plants was a sub-category of GMO foods and therefore that the CRISPR technique would henceforth be regulated in the European Union by their rules and regulations for GMOs. [24]

In February 2020, a US trial safely showed CRISPR gene editing on 3 cancer patients. [25]

Genome engineering

DNA repair after double-stranded break DNA Repair.png
DNA repair after double-stranded break

CRISPR-Cas9 genome editing is carried out with a Type II CRISPR system. When utilized for genome editing, this system includes Cas9, crRNA, and tracrRNA along with an optional section of DNA repair template that is utilized in either non-homologous end joining (NHEJ) or homology directed repair (HDR).

Overview of CRISPR-Cas9 plasmid construction CRISPR overview - en.svg
Overview of CRISPR-Cas9 plasmid construction

Major components

ComponentFunction
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.
sgRNASingle-guide RNAs are a combined RNA consisting of a tracrRNA and at least one crRNA.
Cas9 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 templateDNA 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.

CRISPR-Cas9 often employs a plasmid to transfect the target cells. [26] The main components of this plasmid are displayed in the image and listed in the table. 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. The crRNA must bind only where editing is desired. 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.

Multiple crRNAs and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA). [27] 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. [28] [29]

Overview of the transfection and DNA cleaving by CRISPR-Cas9 (crRNA and tracrRNA are often joined together as a single strand of RNA when designing a plasmid) CRISPR transfection.png
Overview of the transfection and DNA cleaving by CRISPR-Cas9 (crRNA and tracrRNA are often joined together as a single strand of RNA when designing a plasmid)

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. [26] 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. [30] [31]

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). [26]

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. [32]

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. [26] 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.

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. [33] Chemical transfection techniques utilizing lipids have also been used to introduce sgRNAs in complex with Cas9 into cells. [34] 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). [35] [36] [37]

Controlled genome editing

Several variants of CRISPR-Cas9 allow gene activation or genome editing with an external trigger such as light or small molecules. [38] [39] [40] These include photoactivatable CRISPR systems developed by fusing light-responsive protein partners with an activator domain and a dCas9 for gene activation, [41] [42] or by fusing similar light-responsive domains with two constructs of split-Cas9, [43] [44] or by incorporating caged unnatural amino acids into Cas9, [45] or by modifying the guide RNAs with photocleavable complements for genome editing. [46]

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 4-hydroxytamoxifen (4-HT), [38] 4-HT responsive intein-linked Cas9, [47] or a Cas9 that is 4-HT responsive when fused to four ERT2 domains. [48] Intein-inducible split-Cas9 allows dimerization of Cas9 fragments [49] and rapamycin-inducible split-Cas9 system developed by fusing two constructs of split-Cas9 with FRB and FKBP fragments. [50] Other studies have been able to induce transcription of Cas9 with a small molecule, doxycycline. [51] [52] Small molecules can also be used to improve homology directed repair, [53] often by inhibiting the non-homologous end joining pathway. [54] These systems allow conditional control of CRISPR activity for improved precision, efficiency, and spatiotemporal control.

CRISPR screening

The clustered regularly interspaced short palindrome repeats (CRISPR)/Cas9 system is a gene-editing technology that can induce double-strand breaks (DSBs), single-strand nicks, or anywhere guide ribonucleic acids (RNAs) can bind with the protospacer adjacent motif (PAM) sequence. [55] By simple changing sequence of gRNA, Cas9-endonuclease can be delivered to a gene of interest and induce DSBs. [56] 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. [57] [58] 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. [59] 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. [55] 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 separetly. [60] It's 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. [57]

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. [55] 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. [57]

Examples of pooled knock-out libraries, AddGene [61]
LibraryIDSpeciesPIGenes targetedgRNAs per geneTotal gRNAs
Bassik Mouse CRISPR Knockout Library1000000121 — 1000000130MouseBassikVaries (∼23,000 in total)∼10Varies
Mouse Tumor Suppressor Gene CRISPR Knockout Library113584 EFS backbone

113585 TBG backbone

MouseChen56∼4286
Brie mouse genome-wide library73632 (1 plasmid)

73633 (2 plasmid)

MouseDoench and Root19,674478,637
Bassik Human CRISPR Knockout Library101926 — 101934HumanBassikVaries (∼20,500 in total)∼10Varies
Brunello human genome-wide library73179 (1 plasmid)

73178 (2 plasmid)

HumanDoench and Root19,114476,441
Mini-human AsCpf1-based Human Genome-wide Knockout Library130630HumanDraetta16,9773-417,032 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. [62] [63] [64]

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. [65] 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. [65] 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. [66]

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. Chromosomes that have been successfully deleted in vivo using CRISPR techniques include the Y chromosome and X chromosome of adult lab mice and human chromosomes 14 and 21, in embryonic stem cell lines and aneuploid mice respectively. This method might be useful for treating genetic disorders caused by abnormal numbers of chromosomes, such as Down syndrome and intersex disorders. [67]

Successful in vivo genome editing using CRISPR-Cas9 has been shown in numerous model organisms, including Escherichia coli , [68] Saccharomyces cerevisiae , [69] Candida albicans , [70] Caenorhadbitis elegans , [71] Arabidopsis spp., [72] Danio rerio , [73] and Mus musculus . [74] [75] Successes have been achieved in the study of basic biology, in the creation of disease models, [71] and in the experimental treatment of disease models. [76]

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. [77] [78]

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. [79] [80] [81]

CRISPR can be utilized to create human cellular models of disease. [82] 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). [83] 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. [84] 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. [85] Importantly, these disease phenotypes were absent in control organoids of identical genetic background, but lacking the CRISPR modifications. [83]

A similar approach was taken to model long QT syndrome in cardiomyocytes derived from pluripotent stem cells. [86] These CRISPR-generated cellular models, with isogenic controls, provide a new way to study human disease and test drugs.

Biomedicine

CRISPR-Cas technology has been proposed as a treatment for multiple human diseases, especially those with a genetic cause. [87] 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, [88] including cancer, [89] beta-thalassemia, [90] sickle cell disease, [91] hemophilia, [92] cystic fibrosis, [93] Duchenne's muscular dystrophy, [94] Huntington's disease, [95] [96] and heart disease. [97] 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. [98]

CRISPR in the treatment of infection

CRISPR-Cas-based "RNA-guided nucleases" can be used to target virulence factors, genes encoding antibiotic resistance, and other medically relevant sequences of interest. This technology thus represents a novel form of antimicrobial therapy and a strategy by which to manipulate bacterial populations. [99] [100] Recent studies suggest a correlation between the interfering of the CRISPR-Cas locus and acquisition of antibiotic resistance. [101] This system provides protection of bacteria against invading foreign DNA, such as transposons, bacteriophages, and plasmids. This system was shown to be a strong selective pressure for the acquisition of antibiotic resistance and virulence factor in bacterial pathogens. [101]

Therapies based on CRISPR–Cas3 gene editing technology delivered by engineered bacteriophages could be used to destroy targeted DNA in pathogens. [102] Cas3 is more destructive than the better known Cas9. [103] [104]

Research suggests that CRISPR is an effective way to limit replication of multiple herpesviruses. It was able to eradicate viral DNA in the case of Epstein-Barr virus (EBV). Anti-herpesvirus CRISPRs have promising applications such as removing cancer-causing EBV from tumor cells, helping rid donated organs for immunocompromised patients of viral invaders, or preventing cold sore outbreaks and recurrent eye infections by blocking HSV-1 reactivation. As of August 2016, these were awaiting testing. [105]

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. [106] Researchers recently demonstrated the ability to birth live pig specimens after removing these retroviruses from their genome using CRISPR for the first time. [107]

CRISPR and cancer

As of 2016 CRISPR had been studied in animal models and cancer cell lines, to learn if it can be used to repair or thwart mutated genes that cause cancer. [108]

The first clinical trial involving CRISPR started in 2016. It involved removing immune cells from people with lung cancer, using CRISPR to edit out the gene expressed PD-1, then administrating the altered cells back to the same person. 20 other trials were under way or nearly ready, mostly in China, as of 2017. [89]

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. [109]

Knockdown/activation

A dead Cas9 protein coupled with epigenetic modifiers which are used to repress certain genome sequences rather than cutting it all together. Dead-Cas9 potential applications.png
A dead Cas9 protein coupled with epigenetic modifiers which are used to repress certain genome sequences rather than cutting it all together.

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. [106] 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. [5] Conversely, CRISPR-mediated activation (CRISPRa) promotes gene transcription. [110] Cas9 is an effective way of targeting and silencing specific genes at the DNA level. [111] 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. [112]

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. [112]

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 fusobacteria 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. [113] C2c2 has later been renamed to Cas13a to fit the standard nomenclature for Cas genes. [114]

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. [113]

CRISPR-Cas systems can also be employed for editing of micro-RNA and long-noncoding RNA genes in plants. [115]

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. [116] Many recommend that immunization and reversal drives be developed in tandem with gene drives in order to overwrite their effects if necessary. [117] 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. [118] As such, DNA computing would be required.

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. [119]

Prime editing

Prime editing [120] (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. [121] 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. [122] 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. [123] Those three independent pairing events each provide an opportunity to prevent off-target sequences, which significantly increases targeting flexibility and editing precision. [122] Prime editing was developed by researchers at the Broad Institute of MIT and Harvard in Massachusetts. [124] More work is needed to optimize the methods. [124] [123]

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. [125] 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". [126] [127] 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. [128]

In April 2015, Chinese scientists reported results of an attempt to alter the DNA of non-viable human embryos using CRISPR to correct a mutation that causes beta thalassemia, a lethal heritable disorder. [129] [130] The study had previously been rejected by both Nature and Science in part because of ethical concerns. [131] The experiments resulted in successfully changing only some of the intended genes, and had off-target effects on other genes. The researchers stated that CRISPR is not ready for clinical application in reproductive medicine. [131] In April 2016, Chinese scientists were reported to have made a second unsuccessful attempt to alter the DNA of non-viable human embryos using CRISPR – this time to alter the CCR5 gene to make the embryo resistant to HIV infection. [132]

In December 2015, an International Summit on Human Gene Editing took place in Washington under the guidance of David Baltimore. Members of national scientific academies of the US, UK, and China discussed the ethics of germline modification. They agreed to support basic and clinical research under certain legal and ethical guidelines. A specific distinction was made between somatic cells, where the effects of edits are limited to a single individual, and germline cells, where genome changes can be inherited by descendants. Heritable modifications could have unintended and far-reaching consequences for human evolution, genetically (e.g. gene-environment interactions) and culturally (e.g. social Darwinism). Altering of gametocytes and embryos to generate heritable changes in humans was defined to be irresponsible. The group agreed to initiate an international forum to address such concerns and harmonize regulations across countries. [133]

In February 2017, the United States National Academies of Sciences, Engineering, and Medicine (NASEM) Committee on Human Gene Editing published a report reviewing ethical, legal, and scientific concerns of genomic engineering technology. The conclusion of the report stated that heritable genome editing is impermissable now but could be justified for certain medical conditions; however, they did not justify the usage of CRISPR for enhancement. [134]

In November 2018, Jiankui He announced that he had edited two human embryos to attempt to disable the gene for CCR5, which codes for a receptor that HIV uses to enter cells. He said that twin girls, Lulu and Nana, had been born a few weeks earlier. He said that the girls still carried functional copies of CCR5 along with disabled CCR5 (mosaicism) and were still vulnerable to HIV. The work was widely condemned as unethical, dangerous, and premature. [135] An international group of scientists called for a global moratorium on genetically editing human embryos. [136]

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 human embryos by using CRISPR-Cas9 and related techniques. However, researchers were forbidden from implanting the embryos and the embryos were to be destroyed after seven days. [137]

The US has an elaborate, interdepartmental regulatory system to evaluate new genetically modified foods and crops. For example, the Agriculture Risk Protection Act of 2000 gives the United States Department of Agriculture the authority to oversee the detection, control, eradication, suppression, prevention, or retardation of the spread of plant pests or noxious weeds to protect the agriculture, environment, and economy of the US. The act regulates any genetically modified organism that utilizes the genome of a predefined "plant pest" or any plant not previously categorized. [138] In 2015, Yinong Yang successfully deactivated 16 specific genes in the white button mushroom to make them non-browning. Since he had not added any foreign-species (transgenic) DNA to his organism, the mushroom could not be regulated by the USDA under Section 340.2. [139] Yang's white button mushroom was the first organism genetically modified with the CRISPR-Cas9 protein system to pass US regulation. [140]

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. [141] 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. [142] [143]

In China, where social conditions sharply contrast with those of the West, genetic diseases carry a heavy stigma. [144] This leaves China with fewer policy barriers to the use of this technology. [145] [146]

Recognition

In 2012 and 2013, CRISPR was a runner-up in Science Magazine 's Breakthrough of the Year award. In 2015, it was the winner of that award. [106] CRISPR was named as one of MIT Technology Review 's 10 breakthrough technologies in 2014 and 2016. [147] [148] In 2016, Jennifer Doudna and Emmanuelle Charpentier, along with Rudolph Barrangou, Philippe Horvath, and Feng Zhang won the Gairdner International award. In 2017, Doudna and Charpentier were awarded the Japan Prize in Tokyo, Japan for their revolutionary invention of CRISPR-Cas9. In 2016, Charpentier, Doudna, and Zhang won the Tang Prize in Biopharmaceutical Science. [149]

See also

Related Research Articles

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A germline mutation, or germinal mutation, is any detectable variation within germ cells. Mutations in these cells are the only mutations that can be passed on to offspring, when either a mutated sperm or oocyte come together to form a zygote. After this fertilization event occurs, germ cells divide rapidly to produce all of the cells in the body, causing this mutation to be present in every somatic and germline cell in the offspring; this is also known as a constitutional mutation. Germline mutation is distinct from somatic mutation.

Designer baby Genetically modified human embryo

A designer baby is a baby whose genetic makeup has been selected or altered, often to include a particular gene or to remove genes associated with a disease. This process usually involves analysing a wide range of human embryos to identify genes associated with particular diseases and characteristics, and selecting embryos that have the desired genetic makeup; a process known as preimplantation genetic diagnosis. Other potential methods by which a baby's genetic information can be altered involve directly editing the genome – a person's genetic code – before birth. This process is not routinely performed and only one instance of this is known to have occurred as of 2019, where Chinese twins Lulu and Nana were edited as embryos, causing widespread criticism.

CRISPR Family of DNA sequences found in prokaryotic organisms

CRISPR is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes.

Transcription activator-like effector nuclease

Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. The restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in situ, a technique known as genome editing with engineered nucleases. Alongside zinc finger nucleases and CRISPR/Cas9, TALEN is a prominent tool in the field of genome editing.

Genome editing Type of genetic engineering

Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site specific locations.

Genetic engineering techniques overview about the techniques of genetic engineering

Genetic engineering can be accomplished using multiple techniques. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism. The ability to genetically engineer organisms is built on years of research and discovery on how genes function and how we can manipulate them. Important advances included the discovery of restriction enzymes and DNA ligases and the development of polymerase chain reaction and sequencing.

Cas9 Microbial protein found in Streptococcus pyogenes M1 GAS

Cas9 is a 160 kilo dalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids and which is heavily utilized in genetic engineering applications. Its main function is to cut DNA and therefore it can alter a cell's genome.

CRISPR interference genetic perturbation technique

CRISPR interference (CRISPRi) is a genetic perturbation technique that allows for sequence-specific repression of gene expression in prokaryotic and eukaryotic cells. It was first developed by Stanley Qi and colleagues in the laboratories of Wendell Lim, Adam Arkin, Jonathan Weissman, and Jennifer Doudna. Sequence-specific activation of gene expression refers to CRISPR activation (CRISPRa).

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. 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.

Gene drive

A gene drive is a genetic engineering technology that propagates a particular suite of genes throughout a population by altering the probability that a specific allele will be transmitted to offspring from the natural 50% probability. Gene drives can arise through a variety of mechanisms. They have been proposed to provide an effective means of genetically modifying specific populations and entire species.

A protospacer adjacent motif (PAM) is a 2–6-base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The PAM is a component of the invading virus or plasmid, but is not found in the bacterial host genome and hence is not a component of the bacterial CRISPR locus. Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence. PAM is an essential targeting component which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by the CRISPR-associated nuclease.

CRISPR-Cas design tools are computer software platforms and bioinformatics tools used to facilitate the design of guide RNAs (gRNAs) for use with the CRISPR/Cas gene editing system.

Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 or CRISPR/Cpf1 is a DNA-editing technology analogous to the CRISPR/Cas9 system. Cpf1 is an RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. It prevents genetic damage from viruses. Cpf1 genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations. CRISPR/Cpf1 could have multiple applications, including treatment of genetic illnesses and degenerative conditions.

Human germline engineering is the process by which the genome of an individual is edited in such a way that the change is heritable. This is achieved through genetic alterations within the germ cells, or the reproductive cells, such as the egg and sperm. Human germline engineering is a type of genetic modification that directly manipulates the genome using molecular engineering techniques. Aside from germline engineering, genetic modification can be applied in another way, somatic genetic modification. Somatic gene modification consists of altering somatic cells, which are all cells in the body that are not involved in reproduction. While somatic gene therapy does change the genome of the targeted cells, these cells are not within the germline, so the alterations are not heritable and cannot be passed on to the next generation.

CRISPR activation (CRISPRa) is one type of CRISPR tool that use modified versions of dCas9, a mutation of Cas9 without endonuclease activity, with added transcriptional activators on dCas9 or the guide RNAs (gRNAs). Like a standard CRISPR-Cas9 system, dCas9 activation systems rely on similar components such as Cas9 variants for modulation or modification of genes, gRNAs to guide Cas9 to intended targets, and vectors for introduction into cells. However, while a standard CRISPR-Cas9 system relies on creating breaks in DNA through the endonuclease activity of Cas9 and then manipulating DNA Repair mechanisms for gene editing, dCas9 activation systems are modified and employ transcriptional activators to increase expression of genes of interest. Such systems are usable for many purposes including but not limited to, genetic screens and overexpression of proteins of interest.

Off-target genome editing refers to nonspecific and unintended genetic modifications that can arise through the use of engineered nuclease technologies such as: clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9, transcription activator-like effector nucleases (TALEN), meganucleases, and zinc finger nucleases (ZFN). These tools use different mechanisms to bind a predetermined sequence of DNA (“target”), which they cleave, creating a double-stranded chromosomal break (DSB) that summons the cell's DNA repair mechanisms and leads to site-specific modifications. If these complexes do not bind at the target, often a result of homologous sequences and/or mismatch tolerance, they will cleave off-target DSB and cause non-specific genetic modifications. Specifically, off-target effects consist of unintended point mutations, deletions, insertions inversions, and translocations.

Prime editing is a ‘search-and-replace’ genome editing technology in molecular biology by which the genome of living organisms may be modified. The technology directly writes new genetic information into a targeted DNA site. It uses a fusion protein, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. It mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates.

Genome-wide CRISPR-Cas9 knockout screens

Genome-wide CRISPR-Cas9 knockout screens aim to elucidate the relationship between genotype and phenotype by ablating gene expression on a genome-wide scale and studying the resulting phenotypic alterations. The approach utilises the CRISPR-Cas9 gene editing system, coupled with libraries of single guide RNAs (sgRNAs), which are designed to target every gene in the genome. Over recent years, the genome-wide CRISPR screen has emerged as a powerful tool for performing large-scale loss-of-function screens, with low noise, high knockout efficiency and minimal off-target effects.

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