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CRISPR gene editing (pronounced "crisper") is a
genetic engineering Genetic engineering, also called genetic modification or genetic manipulation, is the direct manipulation of an organism's gene In biology, a gene (from ''genos'' "...Wilhelm Johannsen coined the word gene to describe the Mendelian_in ...
technique in
molecular biology Molecular biology is the branch of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, P ...
by which the
genome In the fields of molecular biology Molecular biology is the branch of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, M ...

genome
s of living organisms may be modified. It is based on a simplified version of the bacterial
CRISPR CRISPR () (an acronym for clustered regularly interspaced short palindromic repeats) is a family of DNA Deoxyribonucleic acid (; DNA) is a molecule File:Pentacene on Ni(111) STM.jpg, A scanning tunneling microscopy image of pentacen ...

CRISPR
-
Cas9 Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) is a 160 Dalton (unit), kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized ...
antiviral defense system. By delivering the Cas9
nuclease A nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously affect nick (DNA), single and double-strand break, do ...
complexed with a synthetic
guide RNA Guide RNA (gRNA) is a piece of RNAs that function as guides for RNA- or DNA-targeting Enzyme, enzymes, which they form Protein–ligand complex, complexes with. Very often these enzymes will delete, insert or otherwise alter the targeted RNA or ...
(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 Studies Study or studies may refer to: General * Education **Higher education * Clinical trial * Experiment * Observational study * Research * Study skills, abilities and approaches applied to learning Other * Study (art), a drawing or series ...
'' (in living organisms). The technique is considered highly significant in biotechnology and medicine as it allows for the genomes to be edited ''in vivo'' with extremely high precision, cheaply, and with ease. It can be used in the creation of new medicines, agricultural products, and
genetically modified organisms A genetically modified organism (GMO) is any organism In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecula ...
, or as a means of controlling pathogens and pests. It also has possibilities in the treatment of inherited
genetic diseases A genetic disorder is a health problem caused by one or more abnormalities in the genome In the fields of molecular biology Molecular biology is the branch of biology Biology is the natural science that studies life and li ...
as well as diseases arising from
somatic mutation A somatic mutation is a change in the DNA sequence DNA sequencing is the process of determining the nucleic acid sequence A nucleic acid sequence is a succession of bases signified by a series of a set of five different letters that indicat ...
s such as cancer. However, its use in human germline genetic modification is highly controversial. The development of the technique earned
Jennifer Doudna Jennifer Anne Doudna (; born February 19, 1964) is an American biochemist who has done pioneering work in CRISPR gene editing, and made other fundamental contributions in biochemistry and genetics. She received the 2020 Nobel Prize in Chemistr ...

Jennifer Doudna
and
Emmanuelle Charpentier Emmanuelle Marie Charpentier (born 11 December 1968) is a French French (french: français(e), link=no) may refer to: * Something of, from, or related to France France (), officially the French Republic (french: link=no, République fran ...

Emmanuelle Charpentier
the
Nobel Prize in Chemistry ) , image = Nobel Prize.png , alt = A golden medallion with an embossed image of a bearded man facing left in profile. To the left of the man is the text "ALFR•" then "NOBEL", and on the right, the text (smaller) "NAT•" then "M ...
in 2020. The third researcher group that shared the
Kavli Prize The Kavli Prize was established in 2005 through a joint venture between the Norwegian Academy of Science and Letters The Norwegian Academy of Science and Letters ( no, Det Norske Videnskaps-Akademi, DNVA) is a learned society A learned soc ...
for the same discovery, led by
Virginijus Šikšnys Virginijus Šikšnys (born 26 January 1956) is a Lithuanian biochemist. Biography V. Šikšnys studied organic chemistry at Vilnius University, receiving his Masters in 1978, then moved to Moscow State University, Lomonosov Moscow State University ...
, was not awarded the Nobel prize. Working like genetic scissors, the Cas9 nuclease opens both strands of the targeted sequence of
DNA Deoxyribonucleic acid (; DNA) is a molecule A scanning tunneling microscopy image of pentacene molecules, which consist of linear chains of five carbon rings. A molecule is an electrically Electricity is the set of physical ...

DNA
to introduce the modification by one of two methods. Knock-in mutations, facilitated via
homology directed repair Homology directed repair (HDR) is a mechanism in cells to repair double-strand DNA lesions. The most common form of HDR is homologous recombination Homologous recombination is a type of genetic recombination Genetic recombination (also known a ...
(HDR), is the traditional pathway of targeted genomic editing approaches. This allows for the introduction of targeted . 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. 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 Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homology directe ...
(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 Genome editing, or genome engineering, or gene editing, is a type of genetic engineering Genetic engineering, also called genetic modification or genetic manipulation, is the direct manipulation of an organism's gene In biology, a gene ...
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 ''Streptococcus pyogenes'' is a species of Gram-positive bacteria, Gram-positive, aerotolerant bacteria in the genus ''Streptococcus''. These bacteria are extracellular, and made up of non-motile and non-sporing Coccus, cocci (round cells) that ...

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. The ease with which researchers can insert Cas9 and template RNA in order to
silence Silence is the absence of ambient audible Audible may refer to: * Audible (service), an online audiobook store * Audible (American football), a tactic used by quarterbacks * Hearing, the perception of sound * Audible frequency * Audible ra ...
or cause
point mutation A point mutation or substitution is a genetic mutation where a single nucleotide base is changed, inserted or deleted from a DNA or RNA sequence of an organism's genome. Point mutations have a variety of effects on the downstream protein product ...

point mutation
s at specific
loci Locus (plural loci) is Latin for "place". It may refer to: Entertainment * Locus (comics), a Marvel Comics mutant villainess, a member of the Mutant Liberation Front * Locus (magazine), ''Locus'' (magazine), science fiction and fantasy magazine ...
has proved invaluable to the quick and efficient
mapping Mapping may refer to: * Mapping (cartography), the process of making a map * Mapping (mathematics), a synonym for a mathematical function and its generalizations ** Mapping (logic), a synonym for functional predicate Types of mapping * Animated ...
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. 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 was the AAAS's choice for
Breakthrough of the Year The Breakthrough of the Year is an annual award for the most significant development in scientific research made by the AAAS journal ''Science Science () is a systematic enterprise that builds and organizes knowledge Knowledge is a f ...
in 2015. Many
bioethical Bioethics is the study of the ethical issues Ethics or moral philosophy is a branch of philosophy Philosophy (from , ) is the study of general and fundamental questions, such as those about reason, Metaphysics, existence, Epistemolo ...
concerns have been raised about the prospect of using CRISPR for
germline In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, physiological mechanisms ...
editing, especially in human embryos.


History


Other methods

In the early 2000s, German researchers began developing
zinc finger nuclease Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a nuclease, DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enab ...
s (ZFNs), synthetic proteins whose DNA-binding domains enable them to create double-stranded breaks in DNA at specific points. ZFNs has a higher precision and the advantage of being smaller than Cas9, but ZFNs are not as commonly used as CRISPR-based methods. Sangamo provides ZFNs via industry and academic partnerships but holds the modules, expertise—and patents—for making them. In 2010, synthetic nucleases called
transcription activator-like effector nuclease Transcription activator-like effector nucleases (TALEN) are restriction enzyme A restriction enzyme, restriction endonuclease, or '' restrictase '' is an enzyme Enzymes () are protein Proteins are large s and s that comprise one or ...
s (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 Oligonucleotides are short DNA Deoxyribonucleic acid (; DNA) is a molecule File:Pentacene on Ni(111) STM.jpg, A scanning tunneling microscopy image of pentacene molecules, which consist of linear chains of five carbon rings. A molecule ...
primers). Whereas methods such as
RNA interference RNA interference (RNAi) is a biological process in which RNA Ribonucleic acid (RNA) is a polymer A polymer (; Greek ''wikt:poly-, poly-'', "many" + ''wikt:-mer, -mer'', "part") is a Chemical substance, substance or material consisting ...
(RNAi) do not fully suppress gene function, CRISPR, ZFNs, and
TALENs Transcription activator-like effector nucleases (TALEN) are restriction enzyme A restriction enzyme, restriction endonuclease, or '' restrictase '' is an enzyme Enzymes () are protein Proteins are large s and s that comprise one or ...
provide full irreversible
gene knockout A gene knockout (abbreviation: KO) is a genetic technique in which one of an organism In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemi ...
. CRISPR can also target several DNA sites simultaneously simply by introducing different gRNAs. In addition, the costs of employing CRISPR are relatively low.


Discovery

In 2012
Jennifer Doudna Jennifer Anne Doudna (; born February 19, 1964) is an American biochemist who has done pioneering work in CRISPR gene editing, and made other fundamental contributions in biochemistry and genetics. She received the 2020 Nobel Prize in Chemistr ...

Jennifer Doudna
and
Emmanuelle Charpentier Emmanuelle Marie Charpentier (born 11 December 1968) is a French French (french: français(e), link=no) may refer to: * Something of, from, or related to France France (), officially the French Republic (french: link=no, République fran ...

Emmanuelle Charpentier
published their finding that CRISPR-
Cas9 Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) is a 160 Dalton (unit), kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized ...
could be programmed with RNA to edit genomic DNA, now considered one of the most significant discoveries in the
history of biology The history of biology traces the study of the living world from ancient Ancient history is the aggregate of past events


Patents and commercialization

, SAGE Labs (part of Horizon Discovery group) had
exclusive right In Anglo-Saxon law, an exclusive right, or exclusivity, is a de facto, non-tangible prerogative existing in law (that is, the power (sociology), power or, in a wider sense, right) to perform an action or acquire a benefit and to permit or deny oth ...
s from one of those companies to produce and sell genetically engineered rats and non-exclusive rights for mouse and rabbit models. ,
Thermo Fisher Scientific Thermo Fisher Scientific is an American supplier of scientific instrumentation, reagents and consumables, and software services. Based in Waltham, Massachusetts Waltham ( ) is a city in Middlesex County, Massachusetts, United States, and w ...
had licensed intellectual property from ToolGen to develop CRISPR reagent kits. ,
patent rights NPOV disputes from March 2021 A patent is a Title (property), title that gives its owner the legal right to exclude others from making, using, or selling an invention for a limited period of years in exchange for publishing an sufficiency of di ...
to CRISPR were contested. Several companies formed to develop related drugs and research tools. As companies ramped up financing, doubts as to whether CRISPR could be quickly monetized were raised. 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 The Eli and Edythe L. Broad Institute of MIT and Harvard (pronounced ), often referred to as the Broad Institute, is a biomedical and genomic Genomics is an interdisciplinary field of focusing on the structure, function, evolution, mapping ...
, 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. Shortly after, University of California filed an appeal of this ruling.


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, and in August 2017, the EPO announced its intention to allow CRISPR claims in a patent application that MilliporeSigma had filed. the patent situation in Europe was complex, with MilliporeSigma, ToolGen, Vilnius University, and Harvard contending for claims, along with University of California and Broad. 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 The European Union (EU) is a political and economic union of member states that are located primarily in Europe Europe is a which is also recognised as part of , located entirely in the and mostly in the . It comprises the wester ...

European Union
by their rules and regulations for
GMO A genetically modified organism (GMO) is any organism In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecula ...

GMO
s. In February 2020, a US trial safely showed CRISPR gene editing on three cancer patients. In October 2020, researchers Emmanuelle Charpentier and Jennifer Doudna were awarded the Nobel Prize in Chemistry for their work in this field. They made history as the first two women to share this award without a male contributor. In June 2021, the first, small
clinical trial Clinical trials are experiments or observations done in . Such prospective biomedical or behavioral research studies on are designed to answer specific questions about biomedical or behavioral interventions, including new treatments (such as no ...

clinical trial
of
intravenous Intravenous therapy (abbreviated as IV therapy) is a medical technique that delivers fluids, medications and nutrition directly into a person's vein. The intravenous route of administration is commonly used for rehydration or to provide nutrition ...
CRISPR gene editing in humans concludes with promising results. In September 2021, the first CRISPR-edited food has gone on public sale in Japan. Tomatoes were genetically modified for around five times the normal amount of possibly calming
GABA ''gamma-''Aminobutyric acid, or γ-aminobutyric acid , or GABA , is the chief inhibitory An inhibitory postsynaptic potential (IPSP) is a kind of synaptic potential that makes a postsynaptic neuron less likely to generate an action potent ...

GABA
. CRISPR was first applied in tomatoes in 2014.


Genome engineering

CRISPR-Cas9 genome editing is carried out with a Type II CRISPR system. When utilized for genome editing, this system includes
Cas9 Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) is a 160 Dalton (unit), kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized ...
, crRNA, and tracrRNA along with an optional section of DNA repair template that is utilized in either
non-homologous end joining Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homology directe ...
(NHEJ) or
homology directed repair Homology directed repair (HDR) is a mechanism in cells to repair double-strand DNA lesions. The most common form of HDR is homologous recombination Homologous recombination is a type of genetic recombination Genetic recombination (also known a ...
(HDR).


Major components

CRISPR-Cas9 often employs a
plasmid A plasmid is a small, extrachromosomal DNA Extrachromosomal DNA (abbreviated ecDNA) is any DNA that is found off the chromosome A chromosome is a long DNA molecule with part or all of the genetic material of an organism. Most eukaryo ...
to the target cells. 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). 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.


Alternatives to Cas9

Alternative proteins to Cas9 include the following:


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 motifA 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 ...
(PAM) sequence. The target sequence is 20 bases long as part of each CRISPR locus in the crRNA array. 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 Synthesis or synthesize may also refer to: Science Chemistry and biochemistry *Chemical synthesis, the execution of chemical reactions to form a more complex molecule from chemical precursors **Organic synthesis, the chemical synthesis of or ...
. 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). 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. Properly spaced single-stranded breaks in the host DNA can trigger
homology directed repair Homology directed repair (HDR) is a mechanism in cells to repair double-strand DNA lesions. The most common form of HDR is homologous recombination Homologous recombination is a type of genetic recombination Genetic recombination (also known a ...
, which is less error-prone than the
non-homologous end joining Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homology directe ...
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. 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 Electroporation, or electropermeabilization, is a microbiology Microbiology (from Greek Greek may refer to: Greece Anything of, from, or related to Greece Greece ( el, Ελλάδα, , ), officially the Hellenic Republic, is a country loca ...
of DNA, RNA, or ribonucleocomplexes is a common technique, though it can result in harmful effects on the target cells. Chemical transfection techniques utilizing
lipid In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, physiological mechanis ...
s and peptides have also been used to introduce sgRNAs in complex with Cas9 into cells. 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 ''Lentivirus'' is a genus Genus /ˈdʒiː.nəs/ (plural genera /ˈdʒen.ər.ə/) is a taxonomic rank In biological classification In biology, taxonomy () is the scientific study of naming, defining (Circumscription (taxonomy), cir ...

lentivirus
(LVs),
adenovirus Adenoviruses (members of the family In human society, family (from la, familia) is a group of people related either by consanguinity (by recognized birth) or affinity (by marriage or other relationship). The purpose of families is to ma ...

adenovirus
(AdV), and
adeno-associated virus Adeno-associated viruses (AAV) are small virus A virus is a submicroscopic infectious agent In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemi ...
(AAV).


Controlled genome editing

Several variants of CRISPR-Cas9 allow gene activation or genome editing with an external trigger such as light or small molecules. These include photoactivatable CRISPR systems developed by fusing light-responsive protein partners with an activator domain and a dCas9 for gene activation, or by fusing similar light-responsive domains with two constructs of split-Cas9, or by incorporating caged unnatural amino acids into Cas9, or by modifying the guide RNAs with photocleavable complements for genome editing. 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-HT), 4-HT responsive
intein Protein splicing is an intramolecular reaction of a particular protein in which an internal protein segment (called an intein) is removed from a precursor protein with a ligation of C-terminus, C-terminal and N-terminus, N-terminal external protei ...
-linked Cas9, or a Cas9 that is 4-HT responsive when fused to four ERT2 domains. Intein-inducible split-Cas9 allows dimerization of Cas9 fragments and
rapamycin Sirolimus, also known as rapamycin and sold under the brand name Rapamune among others, is a macrolide compound that is used to coat coronary stents, prevent organ rejection, organ transplant rejection, treat a rare lung disease called lymphangi ...

rapamycin
-inducible split-Cas9 system developed by fusing two constructs of split-Cas9 with FRB and
FKBP FKBP, or FK506 binding protein, is a family of protein Proteins are large biomolecule , showing alpha helices, represented by ribbons. This poten was the first to have its suckture solved by X-ray crystallography by Max Perutz and Sir ...
fragments. Other studies have been able to induce transcription of Cas9 with a small molecule,
doxycycline Doxycycline is a broad-spectrum tetracycline-class antibiotic An antibiotic is a type of antimicrobial An antimicrobial is an agent that kills microorganism A microorganism, or microbe,, ''mikros'', "small") and ''organism ...

doxycycline
. Small molecules can also be used to improve homology directed repair, often by inhibiting the non-homologous end joining pathway. These systems allow conditional control of CRISPR activity for improved precision, efficiency, and spatiotemporal control.


CRISPR screening

The clustered regularly interspaced short palindrome repeats 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. Single-strand nicks can also be induced by Cas9 active-site mutants, also known as Cas9 nickases. By simply changing the sequence of gRNA, the Cas9-endonuclease can be delivered to a gene of interest and induce DSBs. 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. 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. 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. There are two
plasmid A plasmid is a small, extrachromosomal DNA Extrachromosomal DNA (abbreviated ecDNA) is any DNA that is found off the chromosome A chromosome is a long DNA molecule with part or all of the genetic material of an organism. Most eukaryo ...
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. 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 In microbiology, the multiplicity of infection or MOI is the ratio In mathematics, a ratio indicates how many times one number contains another. For example, if there are eight oranges and six lemons in a bowl of fruit, then the ratio of orange ...
(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 The genotype of an organism is its complete set of genetic material. Genotype can also be used to refer to the or variants an individual carries in a particular gene or genetic location. The number of alleles an individual can have in a specific ...
to
phenotype In genetics Genetics is a branch of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular inter ...

phenotype
. 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. 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. 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.


Applications


Disease models

Cas9 genomic modification has allowed for the quick and efficient generation of
transgenic A transgene is a gene In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiol ...
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. 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 '''', one of the first model organisms, have seen further refinement in their resolution with the use of Cas9. 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. CRISPR-Cas9 can be used to edit the DNA of organisms ''in vivo'' and to eliminate individual genes or even entire
chromosome A chromosome is a long DNA molecule with part or all of the genome, genetic material of an organism. Most eukaryotic chromosomes include packaging proteins called histones which, aided by Chaperone (protein), chaperone proteins, bind to and ...

chromosome
s 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 Aneuploidy is the presence of an abnormal number of chromosome A chromosome is a long DNA molecule with part or all of the genome, genetic material of an organism. Most eukaryotic chromosomes include packaging proteins called histones whic ...
mice respectively. This method might be useful for treating genetic disorders caused by abnormal numbers of chromosomes, such as
Down syndrome Down syndrome or Down's syndrome, also known as trisomy 21, is a genetic disorder A genetic disorder is a health problem caused by one or more abnormalities in the genome In the fields of molecular biology and genetics Gen ...
and
intersex Intersex people are individuals born with any of several sex characteristics Sexual characteristics are physical or behavioral traits of an organism (typically of a sexually dimorphic organism) which are indicative of its biological sex. Th ...
disorders. Successful ''in vivo'' genome editing using CRISPR-Cas9 has been shown in numerous model organisms, including ''
Escherichia coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative bacteria, Gram-negative, Facultative anaerobic organism, facultative anaer ...

Escherichia coli
'', ''
Saccharomyces cerevisiae ''Saccharomyces cerevisiae'' () is a species of yeast Yeasts are eukaryotic Eukaryotes () are organism In biology, an organism () is any organic, life, living system that functions as an individual entity. All organisms are ...

Saccharomyces cerevisiae
'', ''
Candida albicans ''Candida albicans'' is an opportunistic pathogenic yeast Yeasts are eukaryotic Eukaryotes () are organism In biology, an organism (from Ancient Greek, Greek: ὀργανισμός, ''organismos'') is any individual contiguous ...

Candida albicans
'', ''
Caenorhabditis elegans ''Caenorhabditis elegans'' () is a free-living transparent nematode The nematodes ( or grc-gre, Νηματώδη; la, Nematoda) or roundworms constitute the phylum Nematoda (also called Nemathelminthes), with plant-parasitic nematodes a ...

Caenorhabditis elegans
'', ''
Arabidopsis ''Arabidopsis'' (rockcress) is a genus in the family Brassicaceae Brassicaceae () or Cruciferae () is a medium-sized and economically important Family (biology), family of flowering plants commonly known as the mustards, the crucifers, or the c ...

Arabidopsis
'' spp., ''
Danio rerio The zebrafish (''Danio rerio'') is a freshwater fish are common freshwater fish throughout temperate Eurasia. Freshwater fish are those that spend some or all of their lives in fresh water, such as river A river is a natural flowing water ...
'', and ''
Mus musculus The house mouse (''Mus musculus'') is a small mammal Mammals (from Latin language, Latin , 'breast') are a group of vertebrate animals constituting the class (biology), class Mammalia (), and characterized by the presence of mammary glands ...
''. Successes have been achieved in the study of basic biology, in the creation of disease models, and in the experimental treatment of disease models. 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. CRISPR simplifies the creation of
genetically modified organisms A genetically modified organism (GMO) is any organism In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecula ...
for research which mimic disease or show what happens when a gene is knocked down or mutated. CRISPR may be used at the
germline In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, physiological mechanisms ...
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. CRISPR can be utilized to create human cellular models of disease. For instance, when applied to human pluripotent stem cells, CRISPR has been used to introduce targeted mutations in genes relevant to
polycystic kidney disease Polycystic kidney disease (PKD or PCKD, also known as polycystic kidney syndrome) is a genetic disorder A genetic disorder is a health problem caused by one or more abnormalities in the genome In the fields of molecular biology and ...
(PKD) and
focal segmental glomerulosclerosis Focal segmental glomerulosclerosis (FSGS), also known as “focal glomerular sclerosis” or “focal nodular glomerulosclerosis,” is a histopathologic finding of scarring (sclerosis) of glomeruli ''Glomerulus'' () is a common term used in anat ...
(FSGS). 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. Kidney organoids with mutations in a gene linked to FSGS developed junctional defects between
podocyte Podocytes are cells Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Closed spaces * Monastic cell, a small room, hut, or cave in which a monk or religious recluse lives * Prison cell, a ro ...
s, the filtering cells affected in that disease. This was traced to the inability of podocytes to form microvilli between adjacent cells. Importantly, these disease phenotypes were absent in control organoids of identical genetic background, but lacking the CRISPR modifications. A similar approach was taken to model
long QT syndrome Long QT syndrome (LQTS) is a condition in which repolarization of the heart after a Cardiac cycle, heartbeat is affected. It results in an increased risk of an cardiac arrhythmia, irregular heartbeat which can result in Syncope (medicine), faintin ...
in
cardiomyocytes Cardiac muscle cells or cardiomyocytes (also known as myocardiocytes or cardiac myocytes) are the muscle cells (myocyte A muscle cell is also known as a myocyte when referring to either a cardiac muscle cell Cardiac muscle cells or cardiomyoc ...
derived from pluripotent stem cells. 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. 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, including cancer,
progeria Progeria is a specific type of progeroid syndrome, also known as Hutchinson-Gilford syndrome. Progeroid syndromes are a group of diseases that cause victims to age faster than usual, leading to them appearing older than they are. Patients born w ...

progeria
, beta-thalassemia,
sickle cell disease Sickle cell disease (SCD) is a group of blood disorder Hematologic diseases are disorders which primarily affect the blood & blood-forming organs. Hematologic diseases include rare genetic disorders, anemia, HIV, sickle cell disease & complica ...
,
hemophilia Haemophilia or hemophilia (from Greek language, Greek αίμα, ''hema'' 'blood', and φιλία, philia 'love of'), is a mostly hereditary, inherited genetic disorder that impairs the body's ability to coagulation, make blood clots, a proces ...
,
cystic fibrosis Cystic fibrosis (CF) is a genetic disorder A genetic disorder is a health problem caused by one or more abnormalities in the genome In the fields of molecular biology and genetics, a genome is all genetic information of an organis ...
, Duchenne's muscular dystrophy,
Huntington's disease Huntington's disease (HD), also known as Huntington's chorea, is a neurodegenerative disease that is mostly Genetic disorder#Autosomal dominant, inherited. The earliest symptoms are often subtle problems with mood or mental abilities. A gener ...
, transthyretin amyloidosis and heart disease. CRISPR has also been used to cure
malaria Malaria is a mosquito-borne infectious disease that affects humans and other animals. Malaria causes symptoms Signs and symptoms are the observed or detectable signs, and experienced symptoms of an illness, injury, or condition. A sign fo ...

malaria
in mosquitos, which could eliminate the vector and the disease in humans. 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 MHC Class II molecules are a class of major histocompatibility complex The major histocompatibility complex (MHC) is a large locus on vertebrate DNA containing a set of closely linked polymorphic genes that code for cell surface proteins essen ...
proteins, which often cause transplant rejection. In addition, clinical trials to cure beta thalassemia and sickle cell disease in human patients using CRISPR-Cas9 technology have shown promising results.


CRISPR in the treatment of infection

CRISPR-Cas-based "RNA-guided nucleases" can be used to target
virulence factors Virulence factors are molecules produced by bacteria Bacteria (; common noun bacteria, singular bacterium) are a type of Cell (biology), biological cell. They constitute a large domain (biology), domain of prokaryotic microorganisms. Typical ...
, genes encoding
antibiotic resistance Antimicrobial resistance (AMR or AR) occurs when microbe A microorganism, or microbe,, ''mikros'', "small") and ''organism In biology Biology is the natural science that studies life and living organisms, including their a ...

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. Recent studies suggest a correlation between the interfering of the CRISPR-Cas locus and acquisition of antibiotic resistance. This system provides protection of bacteria against invading foreign DNA, such as
transposon A transposable element (TE, transposon, or jumping gene) is a DNA sequence DNA sequencing is the process of determining the nucleic acid sequence A nucleic acid sequence is a succession of bases signified by a series of a set of five diff ...
s,
bacteriophage A bacteriophage (), also known informally as a ''phage'' (), is a virus A virus is a submicroscopic infectious agent In biology Biology is the natural science that studies life and living organisms, including their anatomy, ...

bacteriophage
s, and plasmids. This system was shown to be a strong selective pressure for the acquisition of antibiotic resistance and virulence factor in bacterial pathogens. Therapies based on CRISPR–Cas3 gene editing technology delivered by engineered bacteriophages could be used to destroy targeted DNA in pathogens. Cas3 is more destructive than the better known Cas9. 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 The Epstein–Barr virus (EBV), formally called ''Human gammaherpesvirus 4'', is one of the nine known human herpesvirus types in the herpes family, and is one of the most common virus A virus is a submicroscopic infectious agent that ...
(EBV). Anti-herpesvirus CRISPRs have promising applications such as removing cancer-causing EBV from tumor cells, helping rid donated organs for
immunocompromised Immunodeficiency, also known as immunocompromisation, is a state in which the immune system The immune system is a network of biological processes that protects an organism from diseases. It detects and responds to a wide variety of pathoge ...
patients of viral invaders, or preventing outbreaks and recurrent eye infections by blocking
HSV-1 Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), also known by their taxonomical names ''Human alphaherpesvirus 1 ''Human alphaherpesvirus 1'' (HHV-1) is a species of virus in the genus ''Simplexvirus'', subfamily ''Alphaherpesvirinae'', family ...
reactivation. , these were awaiting testing. CRISPR may revive the concept of
transplanting In agriculture Agriculture is the science, art and practice of cultivating plants and livestock. Agriculture was the key development in the rise of sedentary Image:Family watching television 1958.jpg, Exercise trends, Increases in seden ...
animal organs into people.
Retrovirus A retrovirus is a type of virus A virus is a submicroscopic infectious agent In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical ...

Retrovirus
es 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. Researchers recently demonstrated the ability to birth live pig specimens after removing these retroviruses from their genome using CRISPR for the first time.


CRISPR and cancer

CRISPR has also found many applications in developing cell-based immunotherapies. 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, . In 2016, the
United States Food and Drug Administration The United States The United States of America (USA), commonly known as the United States (U.S. or US), or America, is a country Contiguous United States, primarily located in North America. It consists of 50 U.S. state, states, a Wash ...
(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. In November 2020, in mouse animal models, CRISPR was used effectively to treat
glioblastoma Glioblastoma, also known as glioblastoma multiforme (GBM), is the most aggressive type of cancer Cancer is a group of diseases involving abnormal cell growth with the potential to invade or Metastasis, spread to other parts of the body. Th ...
(fast-growing brain tumor) and
metastatic Metastasis is a pathogenic agent's spread from an initial or primary site to a different or secondary site within the host's body; the term is typically used when referring to metastasis by a cancerous tumor. The newly pathological sites, then, ...
ovarian cancer Ovarian cancer is a cancer Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumor A benign tumor is a mass of cells Cell most ...
, as those are two cancers with some of the worst best-case prognosis and are typically diagnosed during their later stages. The treatments have resulted in inhibited tumor growth, and increased survival by 80% for metastatic ovarian cancer and tumor cell
apoptosis Apoptosis (from Ancient Greek Ancient Greek includes the forms of the Greek language Greek ( el, label=Modern Greek Modern Greek (, , or , ''Kiní Neoellinikí Glóssa''), generally referred to by speakers simply as Greek (, ) ...

apoptosis
, inhibited tumor growth by 50%, and improved survival by 30% for glioblastoma.


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. 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 In biology, epigenetics is the study of heritable phenotype changes that do not involve alterations in the DNA sequence. The Ancient Greek, Greek prefix ''wikt:epi-, epi-'' ( "over, outside of, around") in ''epigenetics'' implies features that a ...
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. Conversely, CRISPR-mediated activation (CRISPRa) promotes gene transcription. Cas9 is an effective way of targeting and silencing specific genes at the DNA level. 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. Cas9 was used to carry synthetic
transcription factor In molecular biology Molecular biology is the branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, molecular synthesis, modification, m ...
s 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.


RNA editing

In 2016, researchers demonstrated that CRISPR from an ordinary mouth bacterium could be used to edit
RNA Ribonucleic acid (RNA) is a polymer A polymer (; Greek ''wikt:poly-, poly-'', "many" + ''wikt:-mer, -mer'', "part") is a Chemical substance, substance or material consisting of very large molecules, or macromolecules, composed of many Re ...

RNA
. The researchers searched databases containing hundreds of millions of genetic sequences for those that resembled CRISPR genes. They considered the
fusobacteria Fusobacteria are obligately anaerobic non-sporeforming Gram-negative Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram stain, gram-staining method of bacterial differentiation. They are characteriz ...
''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. C2c2 has later been renamed to Cas13a to fit the standard nomenclature for Cas genes. Many viruses encode their genetic information in RNA rather than DNA that they repurpose to make new viruses.
HIV The human immunodeficiency viruses (HIV) are two species of ''Lentivirus ''Lentivirus'' is a genus Genus (plural genera) is a taxonomic rank Taxonomy (general) is the practice and science of classification of things or concepts, inc ...

HIV
and
poliovirus A poliovirus, the causative agent of polio Poliomyelitis, commonly shortened to polio, is an infectious disease An infection is the invasion of an organism's body Tissue (biology), tissues by Pathogen, disease-causing agents, their multi ...
are such viruses. Bacteria with Cas13 make molecules that can dismember RNA, destroying the virus. Tailoring these genes opened any RNA molecule to editing. CRISPR-Cas systems can also be employed for editing of
micro-RNA A microRNA (abbreviated miRNA) is a small single-stranded non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expressi ...
and long-noncoding RNA genes in plants.


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. Many recommend that immunization and reversal drives be developed in tandem with gene drives in order to overwrite their effects if necessary. 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.


''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 enzyme A restriction enzyme, restriction endonuclease, or '' restrictase '' is an enzyme Enzymes () are protein Proteins are large s and s that comprise one or more long chains of . Proteins perform a vast array of functions within organism ...

restriction enzyme
s. Treatment with Cas9 can deplete abundant rRNA while increasing pathogen sensitivity in RNA-seq libraries.


Prime editing

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


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 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  ...
, including labs in the US, China, and the UK, as well as US biotechnology company OvaScience. 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". 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. In April 2015, Chinese scientists reported results of an attempt to alter the DNA of non-viable
human embryos An embryo is the early stage of development of a multicellular organism. In general, in organism In biology, an organism (from Ancient Greek, Greek: ὀργανισμός, ''organismos'') is any individual contiguous system that embodi ...
using CRISPR to correct a mutation that causes
beta thalassemia Beta thalassemias (β thalassemias) are a group of inherited blood disorders Hematologic diseases are disorders which primarily affect the blood & blood-forming organs. Hematologic diseases include rare genetic disorders, anemia, HIV, sickle cel ...
, a lethal heritable disorder. The study had previously been rejected by both ''
Nature Nature, in the broadest sense, is the natural, physical, material world or universe The universe ( la, universus) is all of space and time and their contents, including planets, stars, galaxy, galaxies, and all other forms of matter an ...
'' and ''
Science Science () is a systematic enterprise that builds and organizes knowledge Knowledge is a familiarity or awareness, of someone or something, such as facts A fact is something that is truth, true. The usual test for a statement of ...
'' in part because of ethical concerns. 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 Reproductive medicine is a branch of medicine concerning the male and female reproductive systems. It encompasses a variety of reproductive conditions, their prevention and assessment, as well as their subsequent treatment and prognosis. Reproduct ...
. 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 C-C chemokine receptor type 5, also known as CCR5 or CD195, is a protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array o ...
gene to make the embryo resistant to
HIV The human immunodeficiency viruses (HIV) are two species of ''Lentivirus ''Lentivirus'' is a genus Genus (plural genera) is a taxonomic rank Taxonomy (general) is the practice and science of classification of things or concepts, inc ...

HIV
infection. In December 2015, an International Summit on Human Gene Editing took place in Washington under the guidance of
David Baltimore David Baltimore (born March 7, 1938) is an American biologist A biologist is a professional who has specialized knowledge in the field of biology, understanding the underlying mechanisms that govern the functioning of biological systems w ...

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 cell A somatic cell (from Ancient Greek Ancient Greek includes the forms of the Greek language Greek ( el, label=Modern Greek Modern Greek (, , or , ''Kiní Neoellinikí Glóssa''), generally referred to by speakers simply as Greek (, ...
s, 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 Social Darwinism refers to various societal practices around the world and defined by scholars in Western Europe and North America in the 1870s that applied biological concepts of natural selection Natural selection is the differential ...
). 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. In February 2017, the United States National Academies of Sciences, Engineering, and Medicine (
NASEM The National Academies of Sciences, Engineering, and Medicine (also known as NASEM or the National Academies) is the collective scientific national academy#REDIRECT National academy A national academy is an organizational body, usually operatin ...
) 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 Genome editing, or genome engineering, or gene editing, is a type of genetic engineering Genetic engineering, also called genetic modification or genetic manipulation, is the direct manipulation of an organism's gene In biology, a gene ...
is impermissible now but could be justified for certain medical conditions; however, they did not justify the usage of
CRISPR CRISPR () (an acronym for clustered regularly interspaced short palindromic repeats) is a family of DNA Deoxyribonucleic acid (; DNA) is a molecule File:Pentacene on Ni(111) STM.jpg, A scanning tunneling microscopy image of pentacen ...

CRISPR
for enhancement. In November 2018, announced that he had edited two human embryos to attempt to disable the gene for
CCR5 C-C chemokine receptor type 5, also known as CCR5 or CD195, is a protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array o ...
, which codes for a receptor that
HIV The human immunodeficiency viruses (HIV) are two species of ''Lentivirus ''Lentivirus'' is a genus Genus (plural genera) is a taxonomic rank Taxonomy (general) is the practice and science of classification of things or concepts, inc ...

HIV
uses to enter cells. He said that twin girls,
Lulu and Nana The He Jiankui affair is a scientific and bioethical controversy concerning the use of genome editing Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or rep ...
, had been born a few weeks earlier. He said that the girls still carried functional copies of CCR5 along with disabled CCR5 (
mosaicism Mosaicism or genetic mosaicism is a condition in multi- cellular organisms in which a single organism possesses more than one genetic line as the result of genetic mutation. This means that various genetic lines resulted from a single fertilized eg ...
) and were still vulnerable to HIV. The work was widely condemned as unethical, dangerous, and premature. An international group of scientists called for a global moratorium on genetically editing human embryos.


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 embryo Human embryonic development, or human embryogenesis, refers to the development and formation of the human embryo. It is characterised by the processes of cell division and cellular differentiation of the embryo that occurs during the early sta ...

human embryo
s 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. 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. 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. Yang's white button mushroom was the first organism genetically modified with the CRISPR-Cas9 protein system to pass US regulation. 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. 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. In China, where social conditions sharply contrast with those of the West, genetic diseases carry a heavy stigma. This leaves China with fewer policy barriers to the use of this technology.


Recognition

In 2012 and 2013, CRISPR was a runner-up in ''Science Magazine'''s
Breakthrough of the Year The Breakthrough of the Year is an annual award for the most significant development in scientific research made by the AAAS journal ''Science Science () is a systematic enterprise that builds and organizes knowledge Knowledge is a f ...
award. In 2015, it was the winner of that award. CRISPR was named as one of ''MIT Technology Review''s 10 breakthrough technologies in 2014 and 2016. In 2016,
Jennifer Doudna Jennifer Anne Doudna (; born February 19, 1964) is an American biochemist who has done pioneering work in CRISPR gene editing, and made other fundamental contributions in biochemistry and genetics. She received the 2020 Nobel Prize in Chemistr ...

Jennifer Doudna
and
Emmanuelle Charpentier Emmanuelle Marie Charpentier (born 11 December 1968) is a French French (french: français(e), link=no) may refer to: * Something of, from, or related to France France (), officially the French Republic (french: link=no, République fran ...

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. In 2020, Charpentier and Doudna were awarded the
Nobel Prize in Chemistry ) , image = Nobel Prize.png , alt = A golden medallion with an embossed image of a bearded man facing left in profile. To the left of the man is the text "ALFR•" then "NOBEL", and on the right, the text (smaller) "NAT•" then "M ...
, the first such prize for an all-female team, "for the development of a method for genome editing."


See also

* CRISPR/Cas Tools * The CRISPR Journal * Eugenics * DRACO * Zinc finger * Gene knockout * Genetics * Glossary of genetics * Human Nature (2019 film), ''Human Nature'' (2019 documentary film) * LEAPER gene editing * RNAi * SiRNA * Surveyor nuclease assay * Synthetic biology


References

{{Authority control Biotechnology Genetic engineering Genome editing