CRISPR / Cas method

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CRISPR / Cas complex with DNA

The CRISPR / Cas method (. From English C lustered R egularly I nterspaced S hort P alindromic R epeats - grouped short palindromic repeats at regular intervals and C RISPR- as sociated - CRISPR-associated protein) is a molecular biological method to DNA targeted to cut and change ( genome editing ) . Genes can be inserted, removed or switched off with the CRISPR / Cas system, and nucleotides in a gene can also be changed. Because of its easy implementation, scalability in terms of different target sequences and low cost, the CRISPR / Cas method is increasingly being used in research.

The scientific basis for the development of the CRISPR / Cas method was laid by the discovery and research of the CRISPR sequences and the associated CRISPR / Cas system in the immune system of various bacteria and archaea . The first scientific documentation on the development and use of the method was published in 2012 by a working group led by Emmanuelle Charpentier and Jennifer Doudna . The scientific journal Science declared the CRISPR method Breakthrough of the Year 2015.

principle

Overview of the three phases of the CRISPR / Cas9 process as a defense mechanism

The CRISPR / Cas method is based on an adaptive antiviral defense mechanism of bacteria, the CRISPR . It is used as a method to cut DNA on an identifiable DNA sequence . In this way, for example, DNA sequences can be removed by two cuts - or another DNA sequence can be inserted at the cut after a cut.

The DNA-cutting enzyme called Cas (from English CRISPR-associated , CRISPR-associated ') binds a specific RNA sequence. This RNA sequence is followed by another RNA sequence that can bind to a DNA with a complementary sequence by base pairing . The RNA serves as a bridge between Cas and the DNA to be cut. By linking the enzyme Cas , the RNA and the DNA, the DNA-cutting enzyme Cas is brought into close proximity to the bound DNA, whereupon the enzyme cuts the (indirectly bound) DNA. In the special case of inserting DNA into the interface, a further DNA is added which has overlapping sequences at both ends for one of the two ends of the interface. The DNA to be inserted is connected to the ends of the interface by the cell's own DNA repair . In a variant of the system ( prime editing ), no DNA to be inserted is necessary for an insertion, since the sequence to be inserted is encoded by an RNA extension.

properties

3D structure of the CRISPR-Cas9 complex
As a molecular tool, CRISPR-Cas9 can target DNA double-strand breaks
The DNA double-strand breaks specifically introduced by CRISPR-Cas9 open the way to genetic manipulation

As ribonucleoproteins , Cas proteins can bind certain RNA sequences. The endonuclease Cas9 (also outdated Cas5, Csn1 or Csx12 ) can bind a certain RNA sequence ( crRNA repeat , sequence GUUUUAGAGCU (A / G) UG (C / U) UGUUUUG) and cut DNA in the immediate vicinity. In bacteria, the crRNA repeat sequence is followed by a crRNA spacer sequence that binds to the target DNA. Both parts are referred to together as crRNA . The crRNA-spacer sequence is used in the function of a variable adapter that is complementary to the target DNA and binds to the target DNA. The crRNA repeat sequence forms an RNA secondary structure and is then bound by Cas9 , whereby a change in the protein folding of Cas9 occurs and the target DNA is bound by the crRNA spacer sequence by base pairing. In the CRISPR / Cas method, the crRNA-spacer sequence is changed in order to bind to a different target DNA. The sequence of the crRNA spacer determines which target DNA sequence is bound.

In addition Cas9 and crRNA the presence of a PAM motif is ( English motif protospacer Adjacent , Contiguous design at the Protospacer ') with the sequence NGG (where N is any nucleic followed by two guanines) in the target DNA is essential for activation of Cas9. The DNA is cut three nucleotides before the PAM. In addition, another RNA is necessary, the tracrRNA , from English. trans-acting CRISPR RNA . The tracrRNA is partially complementary to the crRNA, which is why they bind to each other. The tracrRNA binds to a precursor crRNA , forms an RNA double helix and is converted into the active form by RNase III . The two RNA strands of the crRNA and the tracrRNA can also be accommodated in a single, partially self-hybridizing RNA strand ( sgRNA 'single guide RNA'). These components bind the DNA and cut it by the endonuclease function near the binding site on both strands of the target DNA. In living organisms, a double-stranded DNA cut is followed by a DNA repair. The double-strand break produced is repaired by homology-directed repair (HDR) or by non-homologous end joining (NHEJ).

An alternative method with the same range of possible DNA target sequences is the CRISPR / Cpf1 system and the CRISPR / Cas12b system . As an alternative to the CRISPR / Cas system, transcription activator-like effector nucleases (TALEN) and zinc finger nucleases (ZFN) can be used in some cases. However, these two methods are associated with greater effort to adapt to different target sequences, since a protein design is first necessary to change the binding specificity, while the binding specificity of Cas proteins is based on the use of the bound crRNA or sgRNA and is therefore easier to change . All of these methods are colloquially known as gene scissors .

Differences between Cas9, Cpf1 and Cas12b

property Cas9 Cpf1 Cas12b
structure 2 RNA required or 1 sgRNA 1 RNA required (no tracrRNA ) 2 RNA required or 1 sgRNA
Overhang none (blunt end) sticky end sticky end
interface proximal to the recognition sequence distal to the recognition sequence distal to the recognition sequence
Target sequence G-rich PAM T-rich PAM T-rich PAM
Cell type dividing cells resting cells unknown

The CRISPR / Cas system can also be used to modify RNA by adding a DNA oligonucleotide instead of an RNA oligonucleotide. The effect of suppressing a gene by using CRISPR / Cas has various similarities with that of RNA interference , since short RNA pieces of around 18 to 20 nucleotides mediate binding to the target in both bacterial defense mechanisms .

Adaptation to the target sequence

If, instead of the naturally occurring crRNA spacer sequence, another RNA sequence complementary to a DNA target sequence is added to a crRNA repeat sequence and this crRNA is added to a tracrRNA , Cas9 cuts the DNA near the changed target sequence. The sequence that binds to the target DNA consists of 20 nucleotides, of which the 12 nucleotides adjacent to the PAM are particularly decisive for the binding specificity. The Cas9 with the appropriately modified RNA sequences can cut sequence-specific, double-stranded, partially complementary DNA, whereby targeted deletions can be generated. Changing several DNA target sequences at the same time is known as multiplex genome editing .

Nucleotide substitution

Individual cytidines can be converted into thymidine by a fusion protein from Cas9 without double-stranded endonuclease activity with a cytidine deaminase . Correspondingly , an adenosine can be changed into guanosine with a fusion protein which contains the adenosine deaminase . In both cases, substitution mutations arise . Since this method only changes individual bases without cutting the DNA, this process is known as base editing .

Insertions

Double-strand breaks can significantly increase the frequency of homologous recombination , whereby a sequence-specific cut through Cas9 can also be used to insert DNA sequences (e.g. in the course of gene therapy), provided that the DNA to be subsequently inserted is one at both ends respective target sequence has overlapping sequence.

Introduction into cells

By transformation or transfection from a vector , living beings can be "supplemented" with the CRISPR / Cas system which naturally do not have it, e.g. B. some bacterial strains, baker's yeast , fruit flies , zebrafish , mice and humans . Usually, genes from Cas with nuclear localization signal and sgRNA are inserted into an organism via plasmid, alternatively the complex of Cas9 with an sgRNA can also be introduced into cells.

For genome editing in the germline , electroporation and microinjection are used as methods for introducing CRISPR / Cas9 .

Specificity

Incorrect bonds due to the formation of loops in the target DNA (left) or the sgRNA (right)

When double-strand breaks are generated at undesired locations, undesired mutations can arise, which are referred to as off-target effects. Genes can be disrupted in their function through an unspecific cut. The off-target effects include point mutations (base changes), deletions (removal of DNA sequences), insertions (insertions of DNA sequences), inversions (insertions of DNA sequences in reverse order) and translocations (connections with other DNA sequences) Strands). It has been described that sometimes more than 50% of the cuts were made off-target . The binding of the sgRNA to a target sequence also tolerates base mismatches, which increases the number of possible binding sites to several thousand, which results in experimental and safety problems that are typical of unspecific mutations. Experimental problems are misleading and non- reproducible results. A possible development of cancer is seen as a safety problem . The causes of unspecific cuts can be divided into base mismatches and loop formation (of the sgRNA or the target DNA).

In 2015, a team of Chinese researchers injected 86 human embryos with the correct version of the beta globin gene to correct the defect underlying beta thalassemia; It was found that the correct gene at the correct location could only be detected in four cases, but there was a mosaic in the four locations.

Increase in specificity

To minimize unspecific DNA cuts and the mutagenesis that may result from them , various approaches to increasing the specificity are being investigated, such as the protein design of Cas9 or replacement of the endonuclease activity of Cas9 by combination with other endonucleases. Accordingly, various methods for detecting such mutations have been developed. Computer programs and databases have also been developed to predict off-target effects.

Paired nickases

The mutation from aspartic acid to alanine at position 10 in Cas9 (short: D10A) or from histidine to alanine at position 840 (H840A) inactivates the double-stranded endonuclease function of the bound DNA strand while maintaining the RNA-DNA binding function and a single-stranded endonuclease function . In addition, the specificity is determined by the affinity of the RNA-DNA binding to Cas9. By using two different sgRNA whose sequences binding to the target DNA are slightly offset, two different binding Cas-RNA complexes are created, which can increase the specificity of the section. Two slightly different Cas9-sgRNA complexes with different specificities are formed ( paired nickases ). In addition, the staggered single strand breaks result in sticky ends that facilitate the insertion of a DNA with complementary sticky ends. Single strand breaks are closed by base excision repair and HDR, which produce fewer mutations than repair by NHEJ.

Protein design of Cas9

The mutation D1135E (change from aspartic acid to glutamic acid at position 1135) of SpCas9 changes the binding specificity for the protospacer adjacent motif (PAM) and lowers the number of unspecific cuts. The HF1 mutant of Cas9 also leads to a reduction in unspecific DNA cuts.

dCas9 foci

The combination of Cas9 with inactivated nuclease function (dCas9) with the endonuclease FokI was developed to increase the specificity of the DNA cut. FokI is only active as a homodimer . This reduces the number of unspecific cuts to 1 / 10,000.

BhCas12b v4

The protein Cas12b (old name C2c1 , a Cas of type V, class II) from Bacillus hisashii , abbreviated to BhCas12b, with 1108 amino acids is smaller than SpCas9 (1368 amino acids) and therefore also has a smaller gene, which makes it suitable for use in viral vectors ( especially AAV vectors ). However, the generated wild-type , only of BhCas12b at 37 ° C single-strand breaks . Therefore a mutant of BhCas12b called BhCas12b v4 was developed ( K 846 R / S 893R / E 837 G ), which produces double-strand breaks and less unspecific cuts even at the body temperature of mammals.

dCas9-RT: Prime Editing

A deactivated Cas9 (dCas9) - which can still bind to DNA via an RNA, but without cutting DNA - was combined as a fusion protein with a reverse transcriptase (RT). Reverse transcriptases use an RNA template to make DNA - they are RNA-dependent DNA polymerases . In addition, a prime editing guide RNA (pegRNA) is required, which contains both the crRNA repeat sequence for the binding of Cas9 and an RNA sequence binding to the target DNA as well as an RNA template sequence for changing the DNA sequence (analog a primer containing). In this way, insertions, deletions and all 12 possible point mutations can be carried out without double-strand breaks which are susceptible to mutation and, in the case of an insertion, without the need for a DNA sequence to be inserted.

regulation

The use of anti-CRISPR proteins was proposed for the conditional inhibition of CRISPR / Cas as well as for temporal, local ( cell type-specific ) or cell cycle segment-specific control of the CRISPR-Cas method. Since CRISPR / Cas cuts DNA as long as it is active, a time limit can also limit unspecific cuts that can lead to undesired mutations.

For example, anti-CRISPR proteins are induced at a desired point in time or fusion proteins of anti-CRISPR proteins with light-controlled proteins are used to control the timing of the method. The combination of light-sensitive proteins with anti-CRISPR proteins enables CRISPR-Cas to be activated only during light irradiation, for example the combination of the anti-CRISPR protein AcrIIA4 (an inhibitor of Cas9 ) with the light-sensitive protein LOV2 from Avena sativa ( oat seeds ), which is also called CASANOVA . The local effect can be controlled by using cell-type-specific promoters in front of the Cas and sgRNA genes. In addition, by combining the genes of anti-CRISPR proteins ( AcrIIC1 , AcrIIC3 and AcrIIC1 ) with binding sites for miRNAs that are only found in certain tissues ( liver cells and heart muscle cells ) and there inhibit the formation of the anti-CRISPR protein, a tissue-specific Control for liver and heart muscle cells enables. The use of cell cycle segment-specific promoters (promoters of cyclins ) can limit an effect to a segment in the cell cycle.

Types

There are more than 40 different Cas protein families. The families can be divided into two classes with six types (class I with types I, III and IV and class II with types II, V and VI) and further into more than 30 subtypes. In class I the protein component consists of several proteins in a protein complex (effector complex ), while class II only uses one protein (effector protein). Types I and II bind and cut double-stranded DNA and III-A dsDNA and RNA, while type III-B binds and cuts single-stranded RNA or DNA. In all types, the spacer is formed in bacteria by Cas1 and Cas2 . In types IA and IE, the DNA cut is made by Cas3 , while in type II Cas9 , in type III-A Csm6 and in type III-B Cmr4 causes the cut. Types I and III are structurally related, which suggests a common origin. The helical protein Cas of type III has several β-hairpins that push the double helix of the crRNA and the target RNA apart for the cut at intervals of six nucleotides.

The Cas9 mostly comes from either Streptococcus pyogenes ( SpCas9 ) or Staphylococcus aureus ( SaCas9 ), the coding DNA sequence of SaCas9 being around 1000 base pairs shorter. Cas9 belongs to type II and is being used more and more because the protein component only consists of one protein, which means that cloning and overexpression is less complex. Type I Cas proteins, on the other hand, are protein complexes made up of several small proteins.

Classification

Due to the evolutionary development of pathogens and the resulting variety of pathogens, antiviral defense mechanisms must be able to adapt to the various pathogens. This phenomenon is described in the English-language literature as the evolutionary arms race (German evolutionary arms race ). As a result, CRISPR / Cas systems, as important actors in antiviral defense mechanisms, have also adapted to evolutionary changes. This resulted in an increase in the diversity in terms of gene composition of the cas - operon , the architecture of the CRISPR- gene locus and the gene sequences (including within the Cas-core genes). The Cas core genes are the cas genes cas1 to cas6 , which are conserved within many CRISPR / Cas system variants .

A simple and rational classification of CRISPR / Cas systems proves to be advantageous for the following reasons:

  • Explanation of the origin and evolution of the diversity of CRISPR / Cas systems
  • Tracking and integration of newly discovered CRISPR / Cas system variants
  • coherent annotation of CRISPR / Cas gene loci in microbial genomes

The classification of CRISPR / Cas systems based on the phylogenesis of the Cas1 protein, which has been used since the beginning of CRISPR research, turned out to be problematic after further discoveries of genomes with CRISPR / Cas gene loci, because the organization and phylogenesis of the genes of the effector module was determined by the Phylogenesis of the Cas1 protein differed. An effector module is a group of cas genes that is used to identify genetic material . There is also an adaptation module that also includes cas genes and, with the help of effector proteins, contributes to the selection of protospacers that can be integrated into the bacterial genome. The reason for the phylogenetic deviations is probably the recombination between the effector and adaptation modules ( module shuffling ). Thus it was decided that characteristic features of the effector module became the classification feature of CRISPR / Cas systems.

Classification of CRISPR / Cas systems
class Type target Operon organization 1 Subtype Bacterial strain
I.
I.
DNA
cas6 , SS ( cas11 ) , cas7 , cas5 , cas8a1 , cas3 ' , cas3' ' , cas2 , cas4 , cas1 , cas4 , CRISPR
IA
Archaeoglobus fulgidus , AF1859, AF1870-AF1789
DNA
cas6 , cas8b1 , cas7 , cas5 , cas3 , cas4 , cas1 , cas2 , CRISPR
IB
Clostridium kluyveri , CKL_2758-CKL_2751
DNA
cas3 , cas5 , cas8c , cas7 , cas4 , cas1 , cas2 , CRISPR
IC
Bacillus halodurans , BH0336-BH03342
DNA
cas3 , cas8u2 , cas7 , cas5 , cas6 , cas4 , cas1 , cas2 , CRISPR
IU
Geobacter sulfurreducens , GSU0051-GSU0054, GSU0057-GSU0058
DNA
cas3 ' , cas3' ' , cas10d , cas7 (csc2) , cas5 (csc1) , cas6, cas4, cas1, cas2, CRISPR
ID
Cyanothece sp. , Cyan8802_0527-Cyan8802_0520
DNA
cas3 , cas8e (cse1) , SS (cas11, cse2) , cas7 , cas5 , cas6 , cas1 , cas2 , CRISPR
IE
Escherichia coli K12 , ygcB-ygbF
DNA
cas1 , cas2 , cas3 , cas8f (csy1) , cas5f1 (csy2) , cas7f1 (csy3) , cas6f , CRISPR
IF
Yersinia pseudotuberculosis , YPK_1644-YPK_1649
DNA
cas1 , cas2 , cas3 , cas7f2 , cas5f2 , cas6f , CRISPR
IF
(variant)
Shewanella putrefaciens CN-32 , Sputcn32_1819-Sputcn_32_1823
IV
Plasmids
dlnG (csf4) , cas6-like , cas8-like (csf1) , cas7 (csf2) , cas5 (csf3) , CRISPR
IV-A
Thioalkalivibrio sp. K90mix , TK90_2699-TK90_2703
Plasmids
cas8-like (csf1) , SS (cas11) , cas7 (csf2) , cas5 (csf3) , CRISPR
IV-B
Rhodococcus jostii RHA1 , RHA1_ro10069-RHA1_ro10072
III
DNA / RNA
cas6 , cas10 , SS (cas11) , CAS7 (CSM3) , cas5 (CSM4) , CAS7 (CSM5) , CSM6 , cas1 , cas2 , CRISPR
III-A
Staphylococcus epidermidis , SERP2463-SERP2455
RNA?
cas10 , cas7 (csm3) , cas5 (csx10) , SS (cas11, csm2) , cas7 (csm5) , cas7 (csm5) , all1473 , cas7 (csm5) , CRISPR
III-D
Synechocystis sp. 6803, sll7067-sll7063
DNA / RNA
cas7 (cmr1) , cas7 (cmr6) , cas10 , cas7 (cmr4) , SS (cas11, cmr5) , cas5 (csmr3) , CRISPR
III-C
Methanothermobacter thermautotrophicus , MTH328-MTH323
DNA / RNA
cas7 (cmr1) , cas10 , cas5 (cmr3) , cas7 (cmr4) , SS (cas11, cmr5) , cas6 , cas7 (cmr6) , CRISPR
III-B
Pyrococcus furiosus , PF1131-PF1124
II
II
DNA
cas9 , cas1 , cas2 , cas4 , tracrRNA , CRISPR
II-B
Legionella pneumophila str. Paris , Ipp0160-Ipp0163
DNA
cas9 , cas1 , cas2 , csn2 , tracrRNA , CRISPR
II-A
Streptococcus thermophilus , str0657-str0660
DNA
cas9 , cas1 , cas2 , tracrRNA , CRISPR
II-C
Neisseria lactamica 020-06 , NLA_17660-NLA_17680
DNA
cas9 , tracrRNA , CRISPR , cas4 , cas2 , cas1
II-C
(variant)
Micrarchaeum acidiphilum ARMAN-1 , BK997_03320-BK997_03335
V
DNA
cas12a (cpf1) ,cas4,cas1,cas2,CRISPR
VA
Francisella cf. novicida Fx1 , FNFX1_1431-FNFX_1428
DNA
cas12e (casX) , cas4 , cas1 , cas2 , tracrRNA , CRISPR
VE
Deltaproteobacteria bacterium , A2Z89_08250-A2Z89_08265
DNA
cas12b1 (c2c1) ,cas4,cas1,cas2,tracrRNA,CRISPR
VB
Alicyclobacillus acidoterrestris , N007_06525-N007_06535
DNA
cas4 , cas1 , cas2 , cas12b2 , tracrRNA , CRISPR
VB
(variant)
Planctomycetes bacterium RBG_13_46_10 , A2167_01675-A2167_01685
DNA
cas1 , cas12c (c2c3) , CRISPR
VC
Oleiphilus sp. , A3715_16885-A3715_16890
DNA
cas1 , CRISPR , cas12d (casY)
VD
Bacterium CG09_39_24 , BK003_02070-BK003_02075
?
c2c4 , CRISPR
V-U1
Gordonia otitidis , GOOTI_RS19525
?
c2c8 , CRISPR
V-U2
Cyanothece sp. PCC 8801 , PCC8801_4127
?
c2c5 , CRISPR
V-U5
Cyanothece sp. PCC 8801 , PCC8801_2993-PCC8801_2997
?
c2c10 , CRISPR
V-U3
Bacillus thuringiensis HD-771 , BTG_31928
?
c2c9 , CRISPR
V-U4
Rothia dentocariosa M567 , HMPREF0734_01291
VI
RNA
cas13a (c2c2) ,cas1,cas2,CRISPR
VIA
Leptotrichia shahii , B031_RS0110445
RNA
WYL , cas13d , cas1 , cas2 , CRISPR
VI-D
Ruminococcus bicirculans , RBI_RS12820
RNA?
cas13c (c2c7) , CRISPR
VI-C
Fusobacterium perfoetens , T364_RS0105110
RNA?
cas13b (c2c6) , csx28 , CRISPR
VI-B1
Prevotella buccae , HMPREF6485_RS00335-HMPREF6485_RS00340
RNA?
csx27 , cas13b (c2c6) , CRISPR
VI-B2
Bergeyella zoohelcum , HMPREF9699_02005-HMPREF9699_02006
1The genes marked in bold represent genes of effector complexes of CRISPR / Cas systems of class I and genes of effector proteins of CRISPR / Cas systems of class II.

Applications

Example of use of the CRISPR / Cas system in a plasmid
Blockade of gene expression by a Cas9 mutant ( dCas9 ) in the course of CRISPRi

The CRISPR / Cas system can be used for genome editing (deletions / gene knockout and insertions) and thus also for gene therapy .

However, it could be problematic for human applications that the immune system recognizes the endonuclease Cas9, which is of bacterial origin, as an antigen . Over 80% of all healthy people show both an antibody-based humoral immune response and a cellular immune response based on memory T cells . The reason for this is that most people have come into contact with bacteria such as S. pyogenes or S. aureus at some point in their life and so have been able to form appropriate antibodies and memory cells. Body cells whose genome has been edited with CRISPR / Cas9 can in principle be recognized by cytotoxic T cells and destroyed by apoptosis . The therapeutic effect would therefore no longer exist. This problem is currently (as of 2018) being discussed intensively.

The CRISPR / Cas system is also used to remove the genomes of pathogens causing chronic infectious diseases such as the hepatitis B virus and HIV . The targeted modification of individual genes is used in the characterization of oncogenes and thus in the investigation of tumor development . The CRISPR / Cas system is used to examine the functions of partially unknown genes . It is also used to correct mutations in the generation of induced pluripotent stem cells and embryonic stem cells . Further applications are being investigated.

With the CRISPR / Cas system, bacteriophage- resistant bacterial strains were generated, among other things , and this through an adaptive resistance when adding appropriate RNA in industrially important bacteria, e.g. B. in the dairy or wine industry . A mutated Cas without a functional endonuclease ( dCas9 ) can produce a DNA-binding protein which, among other things, analogously to RNAi, leads to a knockdown of endogenous genes, e.g. B. by transformation with a plasmid from which Cas9 and a CRISPR RNA is transcribed ( CRISPRi ). In the course of CRISPRa, transcription can also be initiated using dCas9 as a fusion protein with an activator at another, determinable location in the genome. Characteristic CRISPR sequences can be used to identify bacterial strains containing CRISPR / Cas ( spoligotyping ). A green fluorescent protein can be used with a Cas mutant as a fusion protein to mark DNA sequences (including repetitive sequences such as telomeres ). The CRISPR / Cas system made it possible to shorten the production time of mice with complex genome changes from up to two years to a few weeks.

Plant breeding

The CRISPR method gives plant breeders the opportunity to change varieties of crops in a lighter, more efficient and more flexible way. Studies are already available for several crops, but there are no CRISPR plants on the market.

Caribou Biosciences , the company founded by Doudna and Charpentier in 2011, entered into a strategic partnership with DuPont Pioneer . Depending on the outcome of the patent dispute, DuPont could therefore obtain the rights to use the method for important crops such as corn, canola and soybean and Caribou for smaller markets such as fruit and vegetables. It is also unclear how the ultimate patent owner will behave with regard to the licensing of the method. In September 2016, the Broad Institute granted Monsanto a (non-exclusive) license to use the method . The license does not apply to Gene Drive , applications in tobacco or the production of sterile plants.

At the same time, there is still a lack of clarity regarding the regulation of CRISPR plants (exclusively with deletions, without insertions) in various countries. The crucial question for the possible future commercial use of CRISPR plants in agriculture is whether these are legally considered as genetically modified plants , provided that something has only been removed from the genome and no transgene has been inserted, since genetically modified plants are very important, especially in the EU are strictly regulated. The US Department of Agriculture announced in April 2016 that it would not regulate two organisms produced using the CRISPR method, a non-browning mushroom and a waxy maize with modified starch content .

Control of insects

Discovery story

See also: CRISPR # discovery and properties

The discovery and research of the CRISPR sequences and the associated CRISPR / Cas system in the immune system of various bacteria and archaea has taken place in several steps since it was first described in 1987. Although the function of CRISPR / Cas was not yet known, it was used at the beginning of the 1990s for so-called spoligotyping (the genome typing of bacterial isolates by recognizing the different spacers within the direct repeat region).

In the early 2000s in particular, the connections between the CRISPR sequences in DNA and the cas genes and their importance in the immune defense of bacteria were identified. As of 2008 it was known that the adaptive CRISPR binds DNA.

In 2011, a working group led by Emmanuelle Charpentier and Jennifer Doudna showed that the CRISPR / Cas system of microorganisms can cut specific DNA targets in vitro. They submitted their scientific work on 8 June 2012 in the journal Science , where it was published on 28 June. At the same time, a working group led by Virginijus Šikšnys was working on the method, which submitted their work to Cell in April 2012 ; however, this was rejected, although the editors of Cell subsequently attributed great importance to the article. In May, Šikšnys and colleagues submitted the paper to the Proceedings of the National Academy of Sciences (PNAS), where it was published online on September 4.

Doudna and Charpentier described how specific sections of the genetic material can be removed from a bacterium. Neuroscientist Feng Zhang from the Massachusetts Institute of Technology later succeeded in not only using the CRISPR method in bacteria, but also in optimizing it for all cells. The director of the Broad Institute and supervisors of Feng Zhang, Eric Lander , wrote in January 2016 article on the proportions of the various scientists to the discovery of the CRISPR / Cas system, which due to an unbalanced representation and a suspected conflict of interest has been criticized.

In 2014, Charpentier and Doudna received the Breakthrough Prize in Life Sciences of 2015, endowed with three million dollars for each winner, for their discovery of the CRISPR / Cas method, and have received numerous other prizes. Within the first five years after publication, the method has become a standard method in laboratories and 2,500 scientific publications have been published about it in these five years.

Patent dispute

Both Doudna and Charpentier (University of California, Berkeley) and Zhang (Broad) applied for basic patents on the CRISPR / Cas method. Doudna and Charpentier filed their application with the United States Patent and Trademark Office (USPTO) in May 2012, Zhang in December 2012, and Zhang filed for an express trial. Zhang was granted patent rights in May 2014. The USPTO justified this decision with the fact that Zhang made the method suitable for all cells. Doudna and Charpentier filed a lawsuit with the USPTO against this decision. The process to clarify the authorship started in January 2016. Broad argues that Berkeley described the method for prokaryotes (bacteria), but not sufficiently for eukaryotes (e.g. mice and human cells). Berkeley argues that moving from prokaryotes to eukaryotes does not require inventive step . In February 2017, the USPTO ruled in Broad's favor and denied the University of California's lawsuit on the grounds that the application of the CRISPR / Cas method described by Doudna and Charpentier to eukaryotes was not obvious.

The European Patent Office (EPO) also has to make decisions in patent disputes. With regard to the Berkeley patent application, the EPO has already argued that the application did not adequately describe the invention because the role of the PAM sequences was not emphasized. Berkeley believes that this role is obvious to professionals. The Broad Institute, on the other hand, has already been awarded several patents for the CRISPR / Cas method, but these have been challenged from several sides. For example, plaintiffs point out that Broad's original patent application mentioned a Rockefeller University scientist as a contributor to the invention, but a later version of the application did not.

Risks

The method is a relatively simple, inexpensive, readily available, precise and efficient technique. In the first few years it was not regulated whether pure deletions, which can also result from random mutagenesis during breeding (but not targeted during breeding), should be assessed as genetic engineering . Critics point out that, for example, international standards and precautionary measures are required to prevent wild growth and abuse. There are also fears of criminal or terrorist applications; the American FBI, for example, is observing more or less private Do-it-Yourself (DIY) "garage hackers" ("bio hackers"). There are bioethical concerns regarding the intervention of the human germline using a CRISPR / Cas method on human germ cells in conjunction with in vitro fertilization as part of gene therapy . In the monograph The revolutionary phenotype , the French-Canadian neurologist Jean-François Gariépy warns against genetic modification in humans. Due to the complexity of genetic modifications, a change in the human genome based on computer programs is inevitable. Based on the RNA world hypothesis , according to which RNA-based forms of life were replaced by DNA-based ones, Gariépy argues that such an approach could in the long term also lead to a revolutionary transformation of the human form of life with unforeseeable consequences. The German Ethics Council warns of "undesirable health consequences" in view of still immature procedures. In addition, there would be unpredictable ethical consequences.

Law

The European Court of Justice (ECJ) ruled on 25 July 2018 that principle also with the CRISPR / Cas method (mutagenesis) worked plants without foreign DNA as genetically modified organisms (GMOs) are to be considered and in principle laid down in the Directive on GMOs Obligations are subject to (Az. C-528/16). The French farmers' union Confédération Paysanne and eight other associations had sued the French government.

The verdict was praised by environmentalists, while natural scientists were critical of the effects, as the removal of DNA would also be achieved by classic methods of breeding, such as treatment with radioactive radiation or with mutagenic chemicals (whose products are not classified as GMOs), however classical methods at random. It is questionable whether research funding will be granted for such projects from now on. In addition, approval is more complex. Research in this regard within the EU is no longer profitable for companies under the new legal framework. Therefore it will no longer take place within the EU. Furthermore, adverse effects on world trade were feared.

literature

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