Patent Application
20230407377
The present invention relates to methods and systems for screening and/or analysing the effect of genetic sequence variants on phenotypic parameters of cells. The genetic variants may be introduced in the genome using a gene editing system such as the CRISPR/Cas9 gene editing system.
In all branches of life science and medicine, a need exists for assays that can determine how genetic sequence variants affect phenotypic parameters of cells, such as proliferation, survival, motility, metabolism or differentiation. A large need exists for example in the field of functional genetic diagnostics, personalized medicine and, personalised drug development. Thus, millions of sequence variants in thousands of genes in millions of patients are nowadays being revealed to doctors by routine patient DNA sequencing. But in the large majority of cases, doctors do not know if the variant is benign or pathogenic, even though this knowledge is essential for diagnosis and clinical management, which could involve drugs tailed to the disease-causing gene (called personalised medicine). Functional genetic diagnosis and testing for drug responsiveness would be the gold standard to solve these questions. Unfortunately, the currently available technologies for this are very laborious, time consuming and expensive and, consequently rarely used in the clinic. As another example, pharma uses huge resources to develop drugs against mutated genes (targeted therapies) or drugs that specifically act on cells with a mutation, but having a target other than the mutated gene (synthetic lethal therapies). Again, the availability of functional genetic model cell systems harbouring desired mutations for drug testing and screening would vastly accelerate the development of such therapies, but the current technologies for generation of genetic model cell systems impede progress. While oncology is the single field with the biggest unmet need for functional genetics, the demand is rapidly on the rise in other major diseases like diabetes/metabolic disorders, inflammation and neurological disease. Furthermore, in humans over 10,000 different monogenetic diseases exist, many of which would benefit tremendously from improved functional genetics methods for diagnosis, treatment and drug development.
The need to determine how a specific genotype impacts cell phenotype, however, is universal in life science and biotech. For instance, functional genetics may determine how genetic variants affect plant cell growth and biosynthesis or response to environmental factors, like drought, heat and pathogens. In bioproduction, functional genetic techniques may be used to improve quantity and quality of products derived from cellular organisms, be it animals, plants, yeast or bacteria.
Gene editing on populations of cells rank as the most powerful and commonly used method to assess the functional consequence of a mutation, reflected by the Nobel prize in chemistry 2020 awarded to the inventors or CRISPR/Cas9 editing. Accordingly, gene editing technologies have during the past decade become the centre of development and innovation in the life sciences, with aims to further investigate how different genotypes influence the phenotype of cells, tissues and organisms. However, a commonly encountered uncertainty in the assessment of the functional cellular effect of an induced mutation is to which extent the experimental design and practice affects the results.
In conventional knock-in experiments using gene-editing technologies, a mutation is introduced in the genome of a cell, the cell is expanded to a clonal cell population, which is finally analysed to determine the effect of the mutation. In this approach, the resulting cell behaviour may be influenced by many factors including transfection toxicities, off-target effects of the editing tool, unwanted selection of traits in the clone expansion step, as well as heterogeneity in the engineered cell population, to name a few. For example, a benign cancer mutation may show apparent loss-of-function effects on cellular fitness due to off-target effects on another genomic locus affecting cell proliferation. Alternatively, the clone(s) selected for analysis may incidentally be slow growing due to clonal variability (heterogeneity) in the cell line studied; another false positive result.
To address these multiple shortcomings, many repeat experiments and individually designed controls are necessary, all of which translates to high labour and material costs, and a lengthy time-to-results. Lengthy time-to-results is also inherent to the process due to the step of cell clone generation. Finally, if the mutation interferes with cell viability, it may not be possible to generate clones for study of the mutation.
There thus exists an urgent need for improved methods that can overcome these limitations.
As outlined above, conventional techniques aiming to elucidate the effects of specific genetic mutations in a cell are associated with a number of drawbacks that include a high propensity for false positive results. Consequently, these methods require appropriate countermeasures that result in a high amount of labour, time and materials being required to assess the effects of each individual mutation. Furthermore, the generation of clonal cell lines for analysis is inherently lengthy and requires that the mutation allows clone expansion.
Interestingly, the present invention provides methods that are accurate, fast, simple, cost-efficient and scalable for introducing one or more mutations of interest in a cell population and determining the effect of the variant on a broad range of cell parameters, such as proliferation and/or survival, within a short timeframe of one to a few weeks for complex cell lines, such as human cells. The use of an internal control containing one or more synonymous mutations at the same, or nearby position as the mutations of interest, and the analysis of hundreds of knock-in cells simultaneously will ensure that the observed differences in cell behaviour are only due to the mutations of interest. In one design of the PCR or next-generation sequencing (NGS) analysis of the introduced mutations, primers are designed to anneal outside the region substantially similar to the oligonucleotides introduced, allowing absolute frequencies to be calculated such that it can be controlled that statistically sufficient numbers of mutant cells underlie the results. A preferred design of the PCR/NGS analysis of the introduced mutations allows that absolute frequencies can be calculated such that it can be controlled that statistically sufficient numbers of mutant cells underlie the results
Another internal control for neutral mutations validates that they are truly neutral and not false negatives due to improper assay functioning. Performing the methods on a cell population eliminates the need for lengthy clone generation and allows for the study of mutations that interfere with clone generation. Finally, a flexible design allows to study the interaction of mutations of interest with cell environmental factors, such as drugs, stresses or pathogens. In this sense, the present methods provide a fast, reliable and versatile indication of what phenotypic effect a mutation of interest will have.
The invention is as defined in the claims.
Herein is provided a method for assessing the effects of a mutation of interest in a cell, said method comprising the steps of:
Also provided herein is a system comprising:
Additionally provided herein is a host cell or host cell population comprising the system as described herein.
Also provided herein is use of a system as described herein in a method for assessing the effects of a mutation of interest in a cell, wherein the method is as described herein.
normalized to the value at Day 2 and are means of 3 independent experiments +/−SD. Two-tailed unpaired t-tests were used to calculate the significance in all cases. NS=not significant. ** indicates p<0.01. **** indicates p<0.0001.
Binding region of a nuclease: the term refers to the region or portion of a target nucleic acid sequence to which a given nuclease actually binds. The CRISPR-Cas binding region comprises an approximately 20 nucleotides long sequence that binds the crRNA by Watson-Crick base pairing. In addition, the Cas protein binds a 2-5 nucleotides long PAM sequence located on the opposite DNA strand and immediately 3′ to the crRNA binding region. The TALEN binding region comprises two 12-16 nucleotides long sequences separated by 5-15 base pairs and located on opposing strands of DNA and that each bind one TALEN of the TALEN dimer. The length of the binding region depends on the number of TALE domains in the individual TALEN monomers. The ZFN binding region comprises two 9-15 nucleotides long sequences separated by 4-6 base pairs and located on opposing strands of DNA and that each bind one ZFN of the ZFN dimer. The length of the binding region depends on the number of zinc finger domains in the individual ZFN monomers.
Cell marker: the term refers to any marker that can be used to characterise a given population or subpopulation of cells, in particular a parameter of interest.
Coding region or coding sequence (CDS): these terms, used herein interchangeably, refer to a portion of DNA or RNA, excluding introns, which results in a protein upon translation.
CRISPR/Cas nuclease: the term refers to members of the CRISPR-Cas family. The prokaryotic adaptive immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) can bind and cleave a target DNA sequence through RNA-guided recognition. According to their molecular architecture, the different members of the CRISPR-Cas system have been classified in two classes: class 1 encompasses several effector proteins, whereas class 2 systems use a single element. The CRISPR-associated proteins (Cas) known to date include Cas9, Cas12a (formerly Cpf1) and Cas13 (formerly C2c2).
Difference between an initial and a subsequent ratio: the term refers to subtracting the value of the initially measured ratio, from the value of the subsequently measured ratio.
For example, if an initial ratio of 1 is measured, and a subsequent ratio of 5 is measured, the difference between the initial and the subsequent ratio is 5-1=4, and therefore positive. In a similar manner, if an initial ratio of 1 is measured, and a subsequent ratio of 0.5 is measured, the difference between the initial and the subsequent ratio is 0.5-1=−0.5, and therefore negative.
Frameshift indel: the term refers to a genetic mutation caused by indels (insertions or deletions) of a number of nucleotides in a DNA sequence that is not divisible by three. This results in a frameshift in the open reading frame of the encoded gene. A change in the frame of the open reading frame often leads to a change in the encoded chain of amino acids or to the introduction of premature stop codons resulting in a shortened and/or non-functional protein product encoded by that gene.
Genotype: the term as used herein refers to an organism comprising a specific set of genes. Thus, two organisms comprising identical genomes are of the same genotype. An organism's genotype in relation to a particular gene is determined by the alleles carried by said organism. In diploid organisms the genotype for a given gene may be AA (homozygous, dominant) or Aa (heterozygous) or aa (homozygous, recessive).
Guide RNA: the term will herein be used interchangeably with “crRNA” and refers to the RNA molecule, which is required for recognition of a target nucleic acid sequence by CRISPR-Cas proteins.
Homologue: a homologue or functional homologue may be any polypeptide that exhibits at least some sequence identity with a reference polypeptide and has retained at least one aspect of the original functionality.
Indel mutation: the term refers to a mutation occurring in a nucleic acid sequence, for example a genomic sequence of an organism, including non-coding regions, coding regions and regulatory sequences such as promoters, which results in insertion and/or deletion of a number of base pairs.
Meganuclease: the term refers to an endodeoxyribonuclease characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs). Meganucleases can be modified to recognise a given target nucleic acid.
Mixed population of cells: the term herein refers to the entire cell population which is contacted with the nuclease and/or the targeting means, and in which it is desired to generate a double-strand break and/or a single-strand break at least in some of the cells. The mixed population of cells therefore typically contains the following types of cells: cells in which no break has been generated and cells in which a break has been generated, and in which either i) perfect repair has occurred and no mutations have been introduced in the target region, ii) an indel mutation has been introduced in the target region, iii) a first oligonucleotide comprising a mutation of interest, preferably a non-silent mutation, preferably a non-synonymous mutation, wherein said mutation preferably lies within the binding region of the nuclease and otherwise identical to, or complementary to the target nucleic acid sequence has been integrated, or copied, or ii) a second oligonucleotide comprising a synonymous mutation, and wherein said synonymous mutation is located in the same position, or close to the position of said mutation of interest, such as within 10 nucleotides of the position of said mutation of interest, and wherein said synonymous mutation preferably lies within the binding region of the nuclease, has been integrated, or copied.
Mutation: the term refers to a mutation occurring in a nucleic acid sequence, for example a genomic sequence, including non-coding regions, coding regions and regulatory sequences such as promoters. Throughout this disclosure, the term “mutation” refers to a change in nucleic acid sequence compared to a reference sequence, for example a wild-type sequence. A mutation may result in a change in the encoded amino acid sequence (non-synonymous mutation) or it may result in no change in the encoded amino acid sequence (synonymous mutation) compared to the amino acid sequence encoded by the reference or wild-type sequence.
Non-silent mutation: the term refers to a mutation occurring in a nucleic acid sequence, for example a genomic sequence of an organism, which results in an observable change in the organism's phenotype or properties. Often a non-silent mutation results in a change of amino acid sequence, efficiency of translation, splicing, a frameshift or a change in a regulatory region, such as a promoter. A non-silent mutation may thus be any mutation in a non-coding region of a genome, or it may be a mutation in a coding region of a genome.
Non-synonymous mutation: the term as used herein refers to a mutation occurring in a nucleic acid sequence, for example a genomic sequence of an organism, which results in a change in the encoded amino acid sequence compared to the amino acid sequence encoded by a reference sequence, typically a wild-type sequence. A non-synonymous mutation thus occurs in coding regions and may be any mutation in a coding region of a genome which changes the amino acid sequence of the translation product, e.g. by encoding a different amino acid (e.g. missense mutations, read-through mutations), by introduction of a premature stop codon (nonsense mutations) and/or by introduction of a frameshift.
Nuclease: the term refers to an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their target nucleic acids. The term encompasses both endonucleases, which effect the break in the nucleic acids from within, and exonucleases, which effect the break from the terminal end(s) of the nucleic acids. The term encompasses deoxyribonucleases acting on DNA, and ribonucleases acting on RNA. Throughout the present disclosure it will be understood that the nucleases can be provided to a cell either as part of a polynucleotide, e.g. a DNA molecule or an RNA molecule, or directly as protein.
Parameter of interest: the term refers to a parameter which is measurable, and which is of interest. In particular, parameters of interest herein refer to parameters associated with the cells that are being investigated, and comprises spatial parameters and temporal parameters, which are further defined below. Examples of relevant parameters of interest include: cell proliferation, cell growth, fitness, cell motility, cell invasiveness, cellular metabolism, cell differentiation, DNA damage, expression levels of pre-defined genes or proteins, resistance to a compound, sensitivity to a compound, production of a compound, anoikis, senescence, apoptosis, DNA methylation, anchorage-independent cell growth, contribution to tumor growth, and protein post-translational modification. For the purpose of the present disclosure, the parameter of interest may be a temporal parameter or a spatial parameter, or a combination of both.
PCR reagents: the term as used herein refers to reagents, which are added to a PCR in addition to a sample and a set of primers. The PCR reagents comprise at least nucleotides and a nucleic acid polymerase. In addition, the PCR reagents may comprise other compounds such as salt(s) and buffer(s).
PCR: the term as used herein refers to a polymerase chain reaction. A PCR is a reaction for amplification of nucleic acids. The method relies on thermal cycling, and consists of cycles of repeated heating and cooling of the reaction to obtain sequential melting and enzymatic replication of said DNA. In the first step, the two strands forming the DNA double helix are physically separated at a high temperature in a process also known as DNA melting. In the second step, the temperature is lowered allowing enzymatic replication of DNA. PCR may also involve incubation at additional temperature in order to enhance annealing of primers and/or to optimize the temperature(s) for replication. In a PCR, the temperature generally cycles between the various temperatures for a number of cycles.
Phenotype: the term as used herein is the composite of the organism's, including single cells' observable characteristics or traits.
Protospacer adjacent motif (PAM): the term refers to the DNA sequence immediately downstream of (3′ to) the DNA sequence targeted by the crRNA of a CRISPR-Cas system and located on the opposite strand. The crRNA of a crRNA-Cas complex is capable of recognizing and hybridizing to a target DNA sequence only if it comprises a PAM, which binds the Cas protein.
Set of primers flanking a target sequence or nucleic acid: the term as used herein refers to a set of two primers flanking a target sequence or a target nucleic acid, so that one primer comprises a sequence identical to a region located 5′ to the target sequence and preferably 5′ to the region homologous to the oligonucleotide comprising the mutation (also referred to as “forward primer”) and one primer comprises a sequence identical to a region located on the opposite DNA strand 3′ to the target sequence and preferably 3′ to the region homologous to the oligonucleotide comprising the mutation (also referred to as “reverse primer”). The “set of primers” can amplify the target sequence when added to a PCR together with a nucleic acid comprising the target sequence region and PCR reagents under conditions allowing amplification of said target sequence.
Silent mutation: the term refers to a mutation occurring in a nucleic acid sequence, for example a genomic sequence of an organism, which does not result in an observable effect on the organism's phenotype or properties. Often a silent mutation is a mutation that does not result in a change of amino acid sequence. A silent mutation may thus be any mutation in a non-coding region of a genome, or it may be a mutation in a coding region of a genome, which does not change the amino acid sequence of the translation product or interfere with translation in other ways.
Synonymous mutation: the term refers to a mutation occurring in a nucleic acid sequence, for example a genomic sequence of an organism, which results in no change in the encoded amino acid sequence. The mutation, unless indicated otherwise, is synonymous compared to the reference sequence, typically a wild-type sequence. A synonymous mutation may thus be any mutation in a coding region of a genome, which does not change the amino acid sequence of the translation product.
Spatial parameter: the term refers to a parameter, in particular a parameter of interest, which is measured and/or monitored spatially in a defined cell population, which may be a total cell population, or a subpopulation of cells. The spatial parameter can, but does not need to, be measured at different time points. The spatial parameter typically reflects a property of the cell population or subpopulation, such as size, biomarker expression, motility, or any parameter that can be measured at a given time point and reflects a spatial or physical property of the cell population or subpopulation. A spatial parameter can thus be measured and/or monitored for a subpopulation of cells resulting for example from FACS sorting, or from isolation using specific antibodies linked to a matrix or for an entire population of cells in e.g. a physical compartment, e.g. an organ or a sub-region of an organ, a tumor or a sub-region of a tumor or on one side of a membrane. Examples of relevant spatial parameters of interest include: cell proliferation, cell growth, fitness, cell motility, cell invasiveness, cellular metabolism, cell differentiation, DNA damage, expression levels of pre-defined genes or proteins, resistance to a compound, sensitivity to a compound, production of a compound, anoikis, senescence, apoptosis, DNA methylation, and protein post-translational modification. The value of the spatial parameter obtained for a given population or subpopulation may in some instances be compared to pre-determined threshold values or to the values for the same parameter obtained in a reference population, which could be another subpopulation or the whole population. However it will be clear from the below description that the value of the spatial parameter for the given population or subpopulation can also be informative on its own, i.e. without comparison to a pre-determined threshold value or the value for a reference population.
Subpopulation of cells: the term refers to a fraction of the mixed population of cells, which comprise at least one single cell. The subpopulations may be physically defined and separated, for example one subpopulation can refer to the cells comprised within a given tumor or sub-region of a tumor or an organ or a sub-region of an organ, while another subpopulation can refer to the cells comprised in healthy tissue or in another organ. As another example, one subpopulation can refer to cells on one side of a cell permeable membrane/matrix, while another subpopulation can refer to cells on the other side. In other instances, the subpopulations are defined on the basis of a value of a spatial or temporal parameter of interest, for example a first subpopulation comprises cells having high values and a second subpopulation comprises cells having low values for the parameter of interest, and such subpopulations may be isolated/separated by FACS. In yet other instances, the subpopulations may be defined by expression of a cell surface protein or epitope, and such subpopulations may be isolated by antibodies linked to a matrix or similar. More than two subpopulations may be defined from a mixed population of cells.
Target nucleic acid: the term will be used interchangeably with the term “target sequence” and herein refers to any nucleic acid sequence within which it is desirable to generate a single-stranded or double-stranded break by the action of a nuclease, for example to introduce a mutation such as a non-silent mutation, a non-synonymous mutation, a silent mutation or a synonymous mutation. Furthermore, the target sequence is preferably a nucleic acid sequence, which can be amplified by PCR technology using primers flanking the target sequence.
Targeting means: the term refers to a moiety or a molecule, which enables a nuclease to recognise its target nucleic acid. For a CRISPR/Cas nuclease, the targeting means refers to the guide RNA or crRNA. For TALENs, the targeting means refers to the TAL effector DNA-binding domains. For ZFNs, the targeting means refers to the zinc finger DNA-binding domains. The targeting means may thus be a moiety of the nuclease (for ZFNs and TALENs), or it may be a different molecule altogether (for CRISPR/Cas systems).
Targeting: the term “targeting” as understood herein refers to the ability of a molecule to identify a nucleotide sequence. For example, an enzyme or a DNA binding domain or molecule may recognise a nucleic acid sequence as a potential substrate and bind to it. Preferably, the targeting is specific.
Temporal parameter: the term refers to a parameter, in particular a parameter of interest, which is measured and/or monitored over time, i.e. the temporal parameter is determined for at least two different time points: an initial time point and a subsequent time point. The ratio of the values measured for the temporal parameter at the initial time point and at the subsequent time point can be calculated to determine a change in ratio. A positive change in ratio for the temporal parameter means that the subsequent value is greater than the initial value, while a negative change in ratio for the temporal parameter means that the subsequent value is smaller than the initial value; no change in ratio indicates that there is no change in the temporal parameter. Examples of relevant temporal parameters of interest include: allele frequencies in a population, cell growth, anchorage-independent cell growth, contribution to tumor growth, fitness, cell motility, cell invasiveness, cellular metabolism, DNA damage, expression levels of pre-defined genes and/or proteins, resistance to a compound, sensitivity to a compound, production of a compound, anoikis, senescence, contact inhibition and apoptosis. The temporal parameter can be measured for an entire population of cells, or for a given subpopulation of cells.
Transcription activator-like effector nuclease (TALEN): these nucleases can be engineered to introduce a break in a target nucleic acid sequence. They are generated by fusing TAL effector DNA-binding domains to a DNA cleavage domain. Engineering of the transcription activator-like effector (TALE) moieties of the TALEN can be performed so that it recognises any given target nucleic acid sequence, which is then cleaved by the DNA cleavage domain. TALENs thus consist of a DNA-binding domain and a DNA cleavage domain, and can introduce a double-strand break in the target nucleic acid. TALENs function as heterodimers composed of two distinct TALENs that bind target nucleic acid sequences on opposing DNA strands.
Zinc finger nuclease: the term refers to enzymes generated by fusing zinc finger DNA-binding domains to a DNA-cleavage domain. Engineering of the zinc finger moieties of the ZFN can be performed so that it recognises any given target nucleic acid sequence, which is then cleaved by the DNA cleavage domain. ZFNs thus consist of a DNA cleavage domain and a DNA-binding domain, and can introduce a double-stranded break in their target nucleic acid. ZFNs function as heterodimers composed of two distinct ZFNs that bind target nucleic acid sequences on opposing DNA strands.
Methods for Assessing the Effects of a Mutation of Interest in a Cell
The present disclosure relates to accurate, fast, simple, cost-efficient methods for assessing the effects of a mutation of interest in a cell. The methods as disclosed herein can be used to determine how genetic variants impact cell proliferation, survival, motility, metabolism, differentiation and/or other cell parameters per se or in response to any environmental stimulus of interest, e.g. drugs, stresses, pathogens and/or nutrients. The methods are scalable and work in any species.
In short, the present methods rely on the introduction, in at least some cells of a cell population, of either a first oligonucleotide comprising a mutation of interest, or a second oligonucleotide comprising a synonymous mutation, where the synonymous mutation does not introduce a change in the encoded amino acid sequence relative to the amino acid sequence encoded by the target nucleic acid sequence or wild-type nucleic acid sequence. Upon generation of a single-stranded or double-stranded break by a nuclease in a target nucleic acid sequence, either the first or the second oligonucleotide are introduced in, or copied into the target nucleic acid sequence. There will also be cells in which the break is perfectly repaired or in which indel mutagenesis occurs, of which the first events will be neutral and the latter events will normally not interfere with the method, but rather will be beneficial for the method in some cases. The population in which the synonymous mutation of the second oligonucleotide is introduced serves as an internal control, which facilitates determining the effect of the mutation of interest introduced via the first oligonucleotide. The resulting population, comprising cells without modification of the target nucleic acid sequence, cells in which an indel mutation has been introduced in the target nucleic acid sequence, cells in which the mutation of interest has been introduced in the target nucleic acid sequence and cells in which a synonymous mutation has been introduced in the target nucleic acid sequence, can then be analysed, and the effects of the mutation of interest assessed as described in detail below. The present methods greatly facilitate assessing the effect of a mutation of interest, and eliminate the need for multiple steps, thereby also reducing the time needed to assess said effect.
In one aspect, the present invention provides a method for assessing the effects of a mutation of interest in a cell, said method comprising the steps of:
The nuclease, the first and/or second oligonucleotide may be introduced, or copied into at least some of the cells in the cell population by any method known in the art. In some embodiments, the nuclease, the first and/or second oligonucleotide are introduced into the cells by electroporation. In some embodiments, the nuclease, the first and/or second oligonucleotide are introduced into the cells by heat shock. In some embodiments, the nuclease, the first and/or second oligonucleotide are introduced into the cells by liposome transfection (lipofection). In some embodiments, the nuclease, the first and/or second oligonucleotide are introduced into the cells by viral delivery. In yet other embodiments, the nuclease, the first and/or second oligonucleotide are introduced into the cells by a non-liposomal transfection reagent such as a FuGENE transfection reagent. In some embodiments, the nuclease may be expressed in the cells and only crRNA and first and/or second oligonucleotide is introduced via any of above-mentioned methods.
In some embodiments, the determined duration is at least 4 hours, such as at least 8 hours, such as at least 12 hours, such as at least 18 hours, such as at least 24 hours, such as at least 48 hours, such as at least 72 hours.
It may also be desirable to wait for longer periods, e.g. if the mutation of interest first has an effect after selective pressure has been applied. Thus, in some embodiments, the determined duration is at least such as at least 1 week, such as at least 2 weeks, such as at least 3 weeks, such as at least 4 weeks, such as at least 2 months, such as at least 4 months, such as at least 6 months, such as at least 8 months, such as at least 10 months, such as at least 12 months, such as at least 1½ year, such as at least 2 years.
In some embodiments, the time-to-result (between steps iii and iv) is at the most 3 weeks, such as at the most 2 weeks, such as at the most 12 days, such as at the most 10 days, such as at the most 8 days, such as at the most one week, such as at the most 6 days, such as at the most 5 days, such as at the most 4 days, such as at the most 3 days, such as at the most 2 days, such as at the most 1 day, such as at the most 12 hours, such as at the most 6 hours.
In some embodiments, the time-to-result (between steps iii and iv) is at the most 2 years, such as at the most 1½ years, such as at the most 12 months, such as at the most 10 months, such as at the most 8 months, such as at the most 6 months, such as at the most 4 months, such as at the most 2 months, such as at the most 4 weeks.
In some embodiments, the time between the initial time point and the subsequent time point is at least 4 hours. In some embodiments, the time between the initial time point and the subsequent time point is at least 8 hours. In some embodiments, the time between the initial time point and the subsequent time point is at least 12 hours. In some embodiments, the time between the initial time point and the subsequent time point is at least 18 hours. In some embodiments, the time between the initial time point and the subsequent time point is at least 24 hours. In some embodiments, the time between the initial time point and the subsequent time point is at least 48 hours. In some embodiments, the time between the initial time point and the subsequent time point is at least 72 hours.
In some embodiments, the time between the initial time point and the subsequent time point is at least 1 week. In some embodiments, the time between the initial time point and the subsequent time point is at least 2 weeks. In some embodiments, the time between the initial time point and the subsequent time point is at least 3 weeks. In some embodiments, the time between the initial time point and the subsequent time point is at least 4 weeks. In some embodiments, the time between the initial time point and the subsequent time point is at least 2 months. In some embodiments, the time between the initial time point and the subsequent time point is at least 4 months. In some embodiments, the time between the initial time point and the subsequent time point is at least 6 months. In some embodiments, the time between the initial time point and the subsequent time point is at least 8 months. In some embodiments, the time between the initial time point and the subsequent time point is at least 10 months. In some embodiments, the time between the initial time point and the subsequent time point is at least 12 months. In some embodiments, the time between the initial time point and the subsequent time point is at least 1½ years. In some embodiments, the time between the initial time point and the subsequent time point is at least 2 years.
In some embodiments, steps iii) and iv) of the method as described herein, and optionally steps A.v) to A.vi) and/or steps B.v) to B.vii), are performed more than once for further predetermined duration(s).
In some embodiments, the target nucleic acid sequence is within a gene, a promoter or an enhancer of a gene, wherein the gene is or is suspected to be an oncogene. In some embodiments, said gene is or is suspected to be a proto-oncogene. In some embodiments, said gene is or is suspected to be a tumor suppressor gene. In some embodiments, said gene is or is suspected to be a gene encoding an enzyme such as an enzyme involved in the production of a compound such as a metabolite, a resistance gene, such as a gene involved in resistance to a compound, a pharmaceutical compound, or a pathogen such as a virus. In some embodiments, said gene is or is suspected to be a gene encoding a protein involved in cellular fitness and/or growth. In some embodiments, said gene is or is suspected to be a gene encoding a protein for any cell function, a microRNA or a long non-coding RNA. The target nucleic acid may be genomic, i.e. comprised in the genome of the cells comprised within the cell population, or it may be extrachromosomal, e.g. on a vector or plasmid or may be comprised in DNA of an infected pathogen or invading organism within another cell.
In some embodiments, step A.v) and/or step B.vi) of the methods as disclosed herein comprises amplifying a region comprising the target nucleic acid sequence, such as by PCR, to produce an amplicon comprising the target nucleic acid sequence, optionally followed by sequencing, such as next-generation sequencing, of said amplicon, in order to determine the ratios of mutation of interest to synonymous mutation.
One advantage of the present methods is that only one set of primers is needed for analysing all types of cells in the mixed cell population. Another advantage is that if the primers are chosen so that they anneal outside the region substantially identical or complementary to the first or second oligonucleotides encompassing the mutations, the same primer set can be used to amplify cells having integrated, or copied the first oligonucleotide, cells having integrated, or copied the second oligonucleotide or cells having integrated or copied none of the oligonucleotides, and instead having performed error-free repair or having obtained an indel mutation at the target region. This critically allows the calculation of the absolute frequencies of introduction of the mutation of interest and the synonymous mutation in the cell population, which in turn allows determination of if the obtained results are statistically significant. This critically also allows for detection of frameshifting indel mutations such that they can serve as internal controls for mutations of interest that appear neutral, by being able to show that the method functioned properly. Thus, similar to the mutation of interest, the ratio of frameshifting indels to WT* can be determined according to a temporal or a spatial parameter to determine if the frameshifting (i.e. knockout/loss-of-function) indel mutation affects the parameter of interest.
Thus, in some embodiments, the mixed population also comprises cells in which the mutation of the first or the second oligonucleotides has been introduced, and/or wherein one or more indel mutations have been introduced in the target nucleic acid sequence.
In some embodiments, step A.v) further comprises a step of determining an initial frequency of cells with an indel in the target nucleic acid sequence which is different from the mutation of the first and the second oligonucleotides, wherein the initial frequency of cells is determined at said initial time point, and determining a subsequent frequency of cells with an indel in the target nucleic acid sequence which is different from the mutation of the first and the second oligonucleotides, wherein the subsequent frequency of cells is determined at said subsequent time point. Said initial frequency of cells with an indel in the target nucleic acid sequence may be determined at the initial time point and/or the subsequent frequency of cells at the subsequent time point.
In some embodiments, step B.vi) further comprises a step of, for each subpopulation, determining a frequency of cells with an indel in the target nucleic acid sequence which is different from the mutation of the first and the second oligonucleotides.
In some embodiments, the step of determining the ratio of cells of step A.v) is performed for only a fraction of the cell population. In some embodiments, the step of determining the ratio of cells of step B.vi) is performed for only a fraction of each subpopulation.
In some embodiments, the initial and the subsequent frequency of cells with an indel is further subdivided into, respectively, an initial and a subsequent frequency of cells with an indel resulting in a frameshift mutation and an initial and a subsequent frequency of cells with an indel not resulting in a frameshift mutation, wherein a subsequent frequency of cells with an indel resulting in frameshift mutation lower or higher than the initial frequency of cells with an indel resulting in frameshift mutation indicates that the frameshift indels are affecting the parameter of interest.
In some embodiments, the frequency of cells with an indel is further subdivided into a frequency of cells with an indel resulting in a frameshift mutation and a frequency of cells with an indel not resulting in a frameshift mutation, wherein a frequency of cells with an indel resulting in frameshift mutation in one subpopulation is substantially different from the frequency of cells with an indel resulting in frameshift mutations in a second subpopulation indicates that the frameshift indels are affecting the cells.
As determining the initial and subsequent ratios of cells in which the mutation of interest has been introduced in the target nucleic acid sequence to cells in which the synonymous mutation has been introduced in the target nucleic acid sequence may be best performed on the same population, it may be desirable to only determine said initial and subsequent ratios on a subset of the population, which serves as a surrogate for the ratio in the complete cell population.
Thus, in some embodiments, the step of determining a ratio of cells in which the mutation of interest has been introduced in the target nucleic acid sequence to cells in which the synonymous mutation has been introduced in the target nucleic acid sequence of step A.v) and/or step B.vi) is performed for only a fraction of the cell population and/or only a fraction of each subpopulation.
In some embodiments, the cell is a vertebrate cell, an invertebrate cell, a plant cell, a yeast cell, a fungal cell or a bacterial cell. In some embodiments, the cell may be part of a library, such as a yeast or a bacterial library, e.g. the Yeast GFP Clone Collection or the E. coli Keio Knockout Collection. These libraries may be used to screen the effect of mutations of interest on e.g. changes in location of specific protein followed by fluorescence microscopy, or e.g. whether the mutations have a larger or a smaller effect depending on if certain proteins are knocked out.
In some embodiments, the cell is a human cell. In some embodiments, the human cell is a primary cell, such as a cell isolated from a patient. Said cell may be isolated from a tumor, e.g. wherein a mutation-of-interest may be introduced in the cell to revert a target sequence, containing a putative cancer-driving mutation, to a wild type sequence in order to diagnose the putative cancer-driving mutation as either contributing to the cancer or not. In some embodiments, the human cell may be isolated from a patient with another genetic disease.
In some embodiments, the methods as described herein are performed in vivo. In yet other embodiments, the methods as described herein are performed in vitro.
Oligonucleotides Useful for the Current Methods
In some embodiments, the first oligonucleotide comprises at least one mutation of interest, such as 1, 2, 3, 4, 5 or more mutations of interest, within, or close to, the binding region of the nuclease, and the second oligonucleotide comprises at least one synonymous mutation, such as 1, 2, 3, 4, 5 or more synonymous mutations, within, or close to, the binding region of the nuclease. In other words, in some embodiments the first and second oligonucleotides differ only in the nature of the mutations they can introduce, but have otherwise the same characteristics, for example the same length and/or GC content.
In some embodiments, the first oligonucleotide comprises at least one mutation of interest, such as 1, 2, 3, 4, 5 or more mutations of interest, within the binding region of the nuclease, and the second oligonucleotide comprises two or more oligonucleotides that each comprise at least one synonymous mutation, such as 1, 2, 3, 4, 5 or more synonymous mutations, within the binding region of the nuclease.
In some embodiments, the first oligonucleotide comprises at least one first mutation, which is a synonymous mutation lying inside the binding region of the nuclease, and further comprises at least one second mutation, which is a non-synonymous mutation of interest lying outside the binding region of the nuclease, and wherein the second oligonucleotide comprises at least one synonymous mutation lying within the same region as the first mutation and further comprises at least one further synonymous mutation lying inside the same region as the second mutation, wherein the first mutation and the synonymous mutation when introduced in the target nucleic acid sequence prevent the nuclease and the targeting means from binding to and/or generating a further SSB or a further DSB in the resulting nucleic acid sequence. In some preferred embodiments, the first mutation and the synonymous mutation are identical.
In some embodiments, the first oligonucleotide and the second oligonucleotide differ only in the location of the mutation of interest. Thus, in some embodiments, the synonymous mutation of said second oligonucleotide is located in the same genomic position as the mutation of interest in said first oligonucleotide.
In some embodiments, said first oligonucleotide consists of or comprises a stretch of nucleotides identical to said second oligonucleotide except for said mutation of interest or wherein said second oligonucleotide consists of or comprises a stretch of nucleotides identical to said first oligonucleotide except for said synonymous mutation.
In some embodiments, the synonymous mutation of said second oligonucleotide is located in a different genomic position from the mutation of interest in said first oligonucleotide. In some embodiments, the synonymous mutation of said second oligonucleotide is located within 20 nucleotides, such as within 19 nucleotides, such as within 18 nucleotides, such as within 17 nucleotides, such as within 16 nucleotides, such as within 15 nucleotides, such as within 14 nucleotides, such as within 13 nucleotides, such as within 12 nucleotides, such as within 11 nucleotides, such as within 10 nucleotides, such as within 9 nucleotides, such as within 8 nucleotides, such as within 7 nucleotides, such as within 6 nucleotides, such as within 5 nucleotides, such as within 4 nucleotides, such as within 3 nucleotides, such as within 2 nucleotides, such as within 1 nucleotide from the genomic position of the mutation of interest in said first oligonucleotide.
In some embodiments, the first and the second oligonucleotides are of different lengths. In some embodiments, the first and the second oligonucleotides are of the same length.
In some embodiments, the synonymous mutation is a single base change compared to the genomic target region. In some embodiments, the mutation of interest is a single base change compared to the genomic target region.
The methods as described herein may also be used for assessing the effects of several different mutations of interest in a cell at the same time. Thus, the present methods may be used for multiplex assays, wherein the effects of multiple mutations are assessed simultaneously in a single experiment.
In some embodiments, the first oligonucleotide is a plurality of first oligonucleotides each comprising a pre-determined mutation of interest and the second oligonucleotide is a plurality of second oligonucleotides each comprising a pre-determined synonymous mutation.
In some embodiments, the first oligonucleotide comprises a plurality of pre-determined mutations of interest and the second oligonucleotide comprises a plurality of pre-determined synonymous mutations. In some embodiments, said second oligonucleotides are otherwise identical to said first oligonucleotides.
In some embodiments, the plurality of mutations is at least 2 different mutations, such as at least 3 different mutations, such as at least 4 different mutations, such as at least different mutations, such as at least 6 different mutations, such as at least 7 different mutations, such as at least 8 different mutations, such as at least 9 different mutations, such as at least 10 different mutations, such as at least 15 different mutations, such as at least 20 different mutations, such as at least 25 different mutations, such as at least different mutations, such as at least 35 different mutations, such as at least 40 different mutations, such as at least 45 different mutations, such as at least 50 different mutations or more.
In some embodiments, one or more of the first oligonucleotide, the second oligonucleotide, the targeting means and the polynucleotide encoding the nuclease are comprised within one or more vectors or plasmids, or within a virus.
In some embodiments, the first and/or the second oligonucleotide is part of the guide RNA capable of hybridizing to the genomic target region. This may be particularly useful if using prime editors to introduce the mutations of the first and/or the second oligonucleotide into cells of the cell population.
Prime editors are fusion proteins between a Cas9 nickase domain and an engineered reverse transcriptase domain. The prime editor may be a fusion of a Streptococcus pyogenes Cas9 nickase domain and a Moloney Murine Leukemia Virus reverse transcriptase domain, such as PE2 (SEQ ID NO: 9) described in Anzalone et al., 2019.
The prime editor protein is targeted to the editing site by a guide RNA, which not only specifies the target site in its spacer sequence, but also encodes the desired mutation in an extension that is typically at the 3′ end of the tracrRNA. Upon target binding, the Cas9 nuclease domain nicks the PAM-containing DNA strand, where upon the newly liberated 3′ end at the target DNA site is used to prime reverse transcription using the extension in the guide RNA as a template. Thereby the mutation is transcribed into the genomic DNA.
In some embodiments, the first oligonucleotide encoding a mutation of interest is part of the guide RNA capable of hybridizing to the genomic target region. In some embodiments, the second oligonucleotide encoding the synonymous mutation is part of the guide RNA capable of hybridizing to the genomic target region. Said first and second oligonucleotides are preferably provided as part of separate guide RNAs capable of hybridizing to the genomic target region.
In some embodiments, one or more of the first oligonucleotide is single-stranded. In some embodiments, one or more of the second oligonucleotide is single-stranded. In some embodiments, one or more of the first oligonucleotide is double-stranded. In some embodiments, one or more of the second oligonucleotide is double-stranded. In some embodiments, one or more of the first oligonucleotide is modified, such as by introduction of one or more phosphorothioate bonds to inhibit oligonucleotide degradation by nucleases. In some embodiments, one or more of the second oligonucleotide is modified, such as by introduction of one or more phosphorothioate bonds to inhibit oligonucleotide degradation by nucleases.
Nucleases Useful for the Current Methods
Any nuclease proficient at generating single- or double-stranded DNA breaks in a target nucleic acid sequence may be used for the described methods. Said nuclease may be directed to cleave the target nucleic acid sequence by a targeting means. The first or the second oligonucleotide can then be introduced at, or copied into, the site of the break.
In some embodiments the cells comprise two or more alleles comprising the target nucleic acid sequence. Without being bound by theory, cells wherein the first or the second oligonucleotide have been introduced at the site of the break of one allele will nearly always have indels introduced in the other allele(s), leading to a deleted, truncated or otherwise non-functional protein being expressed from said other alleles, or the mRNA from said allele may be removed by non-sense mediated mRNA degradation to eliminate protein expression. Such cells will thus effectively only express a likely functional protein from the allele wherein mutation was introduced by the first or second oligonucleotide. This enables assessment of the effects of the mutation without the possibility of complementation from a wild type allele (restoration of the wild type phenotype), such as wherein the wild type gene product is dominant.
In some embodiments, the nuclease comprises or consist of a CRISPR/Cas nuclease and the targeting means comprise or consist of a guide RNA capable of hybridizing to the genomic target region.
In some embodiments, the nuclease is a CRISPR/Cas nuclease, such as the Streptococcus pyogenes Cas9 nuclease of SEQ ID NO: 1, a functional variant thereof which retains nuclease activity, or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. In some embodiments, said nuclease is encoded by a polynucleotide comprising or consisting of SEQ ID NO: 6 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the nuclease is codon-optimised. For example, the nuclease is a human, codon-optimized Streptococcus pyogenes Cas9 (D10A) nickase, such as the human, codon-optimized Streptococcus pyogenes Cas9 (D10A) nickase of SEQ ID NO: 2, a functional variant thereof which retains nickase activity, or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. In some embodiments, said nuclease is encoded by a polynucleotide comprising or consisting of SEQ ID NO: 7 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the nuclease is a Francisella novicida Cas12a nuclease, such as the Francisella novicida Cas12a nuclease of SEQ ID NO: 3, a functional variant thereof which retains nuclease activity, or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. In some embodiments, said nuclease is encoded by a polynucleotide comprising or consisting of SEQ ID NO: 8 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the nuclease is a Francisella novicida Cas12a nuclease, such as MAD7 or a functional variant thereof which retains nuclease activity.
In some embodiments, the nuclease is a Cas9-NG nuclease, such as the Cas9-NG nuclease of SEQ ID NO: 5, a functional variant thereof which retains nuclease activity, or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. In some embodiments, said nuclease is encoded by a polynucleotide comprising or consisting of SEQ ID NO: 4 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
The methods as disclosed herein may readily be used with nuclease prime editors.
In some embodiments, the nuclease is a prime editor, such as the prime editor PE2 of SEQ ID NO: 9, a functional variant thereof which retains nickase and reverse transcriptase activity, or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. In some embodiments, said nuclease is encoded by a polynucleotide comprising or consisting of SEQ ID NO: 10 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, any one of the nucleases as described herein may be codon-optimized for the cell. Thus, in some embodiments, the nuclease is codon-optimized for human cells. In some embodiments, the CRISPR/Cas nuclease, such as the Streptococcus pyogenes Cas9 nuclease of SEQ ID NO: 1 or a functional variant thereof, is codon-optimized for human cells. In some embodiments, the CRISPR/Cas nuclease, such as the Francisella novicida Cas12a nuclease of SEQ ID NO: 3 or a functional variant thereof, or such as MAD7 or a functional variant thereof, is codon-optimized for human cells. In some embodiments, the CRISPR/Cas nuclease, such as the Cas9-NG nuclease of SEQ ID NO: 5 or a functional variant thereof, is codon-optimized for human cells. In some embodiments, the CRISPR/Cas nuclease, such as the prime editor PE2 of SEQ ID NO: 9 or a functional variant thereof, is codon-optimized, for example is codon-optimized for human cells.
In some embodiments, the nuclease and the targeting means comprise or consist of a transcription activator-like effector nuclease (TALEN) consisting of a DNA cleavage domain and a DNA-binding domain. Methods of how to design TALENs in order to cut a given target are known to persons skilled in the art and are also described in Sanjana et al., 2012.
In some embodiments, the nuclease and the targeting means comprise or consist of a zinc finger nuclease consisting of a DNA cleavage domain and a DNA-binding domain. Methods of how to design zinc finger nucleases in order to cut a given target are known to persons skilled in the art and are also described in Carroll et al., 2006 and Wright et al., 2006.
In some embodiments, the nuclease and the targeting means comprise or consist of a meganuclease. Methods of how to design meganucleases in order to cut a given target are known to persons skilled in the art and are also described in Silva et al., 2011.
Selection of Successfully Transfected and/or Genetically Modified Cells
Although a step of selecting cells in the population that have been successfully transfected with the nuclease, or which have successfully integrated either the first or the second oligonucleotide in the target nucleic acid, is not required for the presently disclosed methods, it may nevertheless be desirable to include such a step. Such a step may serve to reduce the background or noise pertaining to amplification errors of the target sequence in non-transfected cells.
Thus, in some embodiments, step ii) of the method as described herein further comprises selecting the cells in which the nuclease or the polynucleotide encoding said nuclease has been introduced, thereby obtaining a subpopulation of cells enriched in cells in which the mutation of interest and/or the synonymous mutation has been introduced in the target nucleic acid sequence.
In yet other embodiments, step ii) of the method as described herein further comprises selecting the cells in which one of the first oligonucleotide or the second oligonucleotide has been introduced, thereby obtaining a mixed population of cells comprising cells in which the mutation of interest has been introduced in the target nucleic acid sequence, and cells in which the synonymous mutation has been introduced in the target nucleic acid sequence.
Said selection step may be performed by methods known in the art, such as by co-expression of a resistance gene together with the nuclease, e.g. from a transfected plasmid, and adding to the cell growth medium the compound that the cells become resistant to by expressing said gene, or such as by including a fluorescent tag on either the nuclease or either of the oligonucleotides, whereby the cells may be cell sorted for the fluorescent signal, e.g. by FACS.
Parameters for Assessment of the Effects of the Mutation of Interest in a Cell
The disclosed methods are useful for assessing the effects of mutations on a broad range of cell parameters. These parameters may be classified as being a temporal parameter, i.e. related to a change over time, and/or a spatial parameter, i.e. related to a specific location in physical space.
In some embodiments the parameter of interest is a cellular response to a compound or an external stimulus, such as temperature, drought or pressure, and the medium of step iii) of the present methods comprises said compound or step iii) additionally comprises subjecting the cell population to the external stimulus, and the cellular response is a temporal parameter or a spatial parameter. In some embodiments, the compound is a therapeutic agent or a candidate therapeutic agent, a virus or a viral agent, a pathogen, an active agent, a metabolite or a cell signaling molecule. In some embodiments, the external stimulus is a physical stimulus. In some embodiments the external stimulus is heat. In some embodiments the external stimulus is cold. In some embodiments the external stimulus is drought. In some embodiments the external stimulus is UV radiation.
Temporal Parameters
In some embodiments, the parameter of interest is a temporal parameter, and the method for assessing the effects of a mutation of interest in a cell comprises the steps of:
In some embodiments, the temporal parameter is cell proliferation. In some embodiments, the temporal parameter is cell growth. In some embodiments, the temporal parameter is anchorage-independent cell growth. In some embodiments, the temporal parameter is contribution to tumor growth. In some embodiments, the temporal parameter is fitness. In some embodiments, the temporal parameter is cell motility. In some embodiments, the temporal parameter is cell invasiveness. In some embodiments, the temporal parameter is cellular metabolism. In some embodiments, the temporal parameter is DNA damage. In some embodiments, the temporal parameter is expression levels of pre-defined genes and/or proteins. In some embodiments, the temporal parameter is resistance to a compound. In some embodiments, the temporal parameter is sensitivity to a compound. In some embodiments, the temporal parameter is production of a compound. In some embodiments, the temporal parameter is anoikis. In some embodiments, the temporal parameter is senescence. In some embodiments, the temporal parameter is contact inhibition. In some embodiments, the temporal parameter is apoptosis.
In some embodiments, the parameter of interest is a temporal parameter of interest, and if the initial ratio of cells in which the mutation of interest has been introduced in the target nucleic acid sequence to cells in which the synonymous mutation has been introduced in the target nucleic acid sequence, is lower than the subsequent ratio of cells in which the mutation of interest has been introduced in the target nucleic acid sequence to cells in which the synonymous mutation has been introduced in the target nucleic acid sequence, the mutation is characterised as a mutation having a positive effect on the temporal parameter.
In some embodiments, the parameter of interest is a temporal parameter of interest, and if the initial ratio of cells in which the mutation of interest has been introduced in the target nucleic acid sequence to cells in which the synonymous mutation has been introduced in the target nucleic acid sequence, is greater than the subsequent ratio of cells in which the mutation of interest has been introduced in the target nucleic acid sequence to cells in which the synonymous mutation has been introduced in the target nucleic acid sequence, the mutation is characterised as a mutation having a negative effect on the temporal parameter.
In some embodiments, the parameter of interest is a temporal parameter of interest, and if the initial ratio of cells in which the mutation of interest has been introduced in the target nucleic acid sequence to cells in which the synonymous mutation has been introduced in the target nucleic acid sequence, is substantially the same as the subsequent ratio of cells in which the mutation of interest has been introduced in the target nucleic acid sequence to cells in which the synonymous mutation has been introduced in the target nucleic acid sequence, the mutation is characterised as a mutation having no effect on the temporal parameter.
As mentioned herein above, the parameter of interest may be a cellular response to a compound or an external stimulus, such as temperature, drought or pressure, and the medium of step iii) of the disclosed methods comprises said compound or step iii) additionally comprises subjecting the cell population to the external stimulus. The effect of the mutation of interest on the parameter can then be determined by exposing the cells to conditions for assessing the parameter, for example the compound or the external stimulus.
In some embodiments, the cellular response is monitored as a temporal parameter, and if the initial ratio of cells is lower than the subsequent ratio of cells, it indicates a positive effect of the mutation of interest on the cellular response to the compound. In some embodiments, the cellular response is monitored as a temporal parameter, and if the initial ratio of cells is greater than the subsequent ratio of cells, it indicates a negative effect of the mutation of interest on the cellular response to the compound. In some embodiments, the cellular response is monitored as a temporal parameter, and if the initial and the subsequent ratio of the cells are substantially the same, it indicates no effect of the mutation of interest on the cellular response to the compound.
Spatial Parameters
In some embodiments, the parameter of interest is a spatial parameter, and the method for assessing the effects of a mutation of interest in a cell comprises the steps of:
In some embodiments, the spatial parameter is cell proliferation. In some embodiments, the spatial parameter is cell growth. In some embodiments, the spatial parameter is fitness. In some embodiments, the spatial parameter is cell motility. In some embodiments, the spatial parameter is cell invasiveness. In some embodiments, the spatial parameter is cellular metabolism. In some embodiments, the spatial parameter is cell differentiation. In some embodiments, the spatial parameter is DNA damage. In some embodiments, the spatial parameter is expression levels of pre-defined genes and/or proteins. In some embodiments, the spatial parameter is resistance to a compound. In some embodiments, the spatial parameter is sensitivity to a compound. In some embodiments, the spatial parameter is production of a compound. In some embodiments, the spatial parameter is anoikis. In some embodiments, the spatial parameter is senescence. In some embodiments, the spatial parameter is apoptosis. In some embodiments, the spatial parameter is DNA methylation. In some embodiments, the spatial parameter is protein post-translational modification.
Spatial parameters may be measured at certain spatial locations of interest, e.g. a subpopulation present on only one side of a cell-permeable membrane, a subpopulation of physically isolated cells that have been FACS-sorted for expression of a cell marker at a higher level compared to a reference cell population, or a specific organ in a test animal having received a xenograft of a transfected cell population, and the ratio of cells in which the synonymous mutation has been introduced in the target nucleic acid sequence to cells in which the mutation of interest has been introduced in the target nucleic acid sequence is then measured in the subpopulation present at said spatial location of interest and compared to the same measurement in a subpopulation present at a reference location. The subpopulation of step B.v) may thus also be defined or separated from other subpopulations on the basis of the value of a certain spatial parameter, e.g. presence on one side of a cell-permeable membrane, expression of a cell marker at a higher level compared to a reference cell population that can be measured by e.g. FACS, or presence in a specific organ in an animal having received a xenograft of a transfected cell population.
Thus, in some embodiments, step B.v) of the disclosed methods comprises defining the subpopulations on the basis of a cell property, such as the ability of the cells to migrate or invade, optionally wherein the cell property is the spatial parameter of interest.
In some embodiments, each subpopulation of step B.v) and/or each reference population of the disclosed methods comprises one or more cells, such as a single cell or a plurality of cells.
In some embodiments, step B.v) of the methods as disclosed herein comprises defining at least one subpopulation of cells and a reference subpopulation of cells from the mixed population on the basis of the spatial parameter of interest, and if the ratio of cells in which the mutation of interest has been introduced in the target nucleic acid sequence to cells in which the synonymous mutation has been introduced in the target nucleic acid sequence in said subpopulation is greater than the ratio of cells in which the mutation of interest has been introduced in the target nucleic acid sequence to cells in which the synonymous mutation has been introduced in the target nucleic acid sequence in a reference subpopulation, the mutation is characterised as a mutation having a positive effect on the spatial parameter.
In some embodiments, step B.v) of the methods as disclosed herein comprises defining at least one subpopulation of cells and a reference subpopulation of cells from the mixed population on the basis of the spatial parameter of interest, and if the ratio of cells in which the mutation of interest has been introduced in the target nucleic acid sequence to cells in which the synonymous mutation has been introduced in the target nucleic acid sequence in said subpopulation is lower than the ratio of cells in which the mutation of interest has been introduced in the target nucleic acid sequence to cells in which the synonymous mutation has been introduced in the target nucleic acid sequence in a reference subpopulation, the mutation is characterised as a mutation having a negative effect on the spatial parameter.
In some embodiments, step B.v) of the methods as disclosed herein comprises defining at least one subpopulation of cells and a reference subpopulation of cells from the mixed population on the basis of the spatial parameter of interest, and if the ratio of cells in which the mutation of interest has been introduced in the target nucleic acid sequence to cells in which the synonymous mutation has been introduced in the target nucleic acid sequence in said subpopulation is substantially the same as the ratio of cells in which the mutation of interest has been introduced in the target nucleic acid sequence to cells in which the synonymous mutation has been introduced in the target nucleic acid sequence in a reference subpopulation, the mutation is characterised as a mutation having no effect on the spatial parameter.
In some embodiments, step B.v) further comprises spatially separating said at least one subpopulation from the reference subpopulation.
In some embodiments, step B.v) of the disclosed methods comprises defining at least one subpopulation of cells and a reference subpopulation of cells from the mixed population on the basis of the spatial parameter of interest and spatially separating said at least one subpopulation from the reference subpopulation, and if the ratio of cells in said subpopulation is greater than in said reference subpopulation, the mutation is characterised as a mutation having a positive effect on the spatial parameter, the mutation is characterised as a mutation having a positive effect on the spatial parameter.
In some embodiments, step B.v) of the methods as disclosed herein comprises defining at least one subpopulation of cells and a reference subpopulation of cells from the mixed population on the basis of the spatial parameter of interest and spatially separating said at least one subpopulation from the reference subpopulation, and if the ratio of cells in said subpopulation is lower than the ratio of said reference subpopulation, the mutation is characterised as a mutation having a negative effect on the spatial parameter, the mutation is characterised as a mutation having a negative effect on the spatial parameter.
In some embodiments, step B.v) of the disclosed methods comprises defining at least one subpopulation of cells and a reference subpopulation of cells from the mixed population on the basis of the spatial parameter of interest and spatially separating said at least one subpopulation from the reference subpopulation, and if the ratio of cells in said subpopulation is substantially the same as the ratio of cells in said reference subpopulation, the mutation is characterised as a mutation having no effect on the spatial parameter.
As mentioned herein above, the parameter of interest may be a cellular response to a compound or an external stimulus, and the medium of step iii) of the disclosed methods comprises said compound.
In some embodiments, the cellular response is monitored as a spatial parameter, wherein for each subpopulation, a ratio of said subpopulation greater than the ratio of a reference subpopulation indicates that the mutation has a positive effect on the cellular response to the compound.
In some embodiments, the cellular response is monitored as a spatial parameter, wherein for each subpopulation a ratio of said subpopulation less than the ratio of a reference subpopulation indicates that the mutation has a negative effect on the cellular response to the compound.
In some embodiments, the cellular response is monitored as a spatial parameter, wherein for each subpopulation a ratio of said subpopulation substantially the same as the ratio of a reference subpopulation indicates that the mutation has no effect on the cellular response to the compound.
To define the value of the parameter of interest, it may be desirable to compare with a reference population, such as a population that has not been transfected with any of the oligonucleotides as described herein above in the section ‘Oligonucleotides useful for the current methods’. Said reference population may also be cells on one side of a cell-permeable membrane, for example cells may be plated on one side of a cell-permeable membrane, and some cells of the cell population may display high invasiveness and/or motility and translocate through the membrane to the other side—in this case the cell population on the side in which the cells were originally plated may be used as the reference population. The reference population may also be an organ in an animal having received a xenograft of transfected cells, e.g. if the parameter of interest relates to which cells have migrated from the xenograft to the liver, cells from another organ, such as the spleen or lungs, may be used as the reference population.
Thus, in some embodiments, the spatial parameter of interest has a value of interest defined by comparison with a reference value of the same spatial parameter in a reference population. The values obtained for a reference population (e.g. side scatter as measured by FACS) may define a value of interest (e.g. what values for side scatter as measured by FACS would be of interest when compared to the reference population). The reference value of the same spatial parameter in a reference population may thus define a cutoff value for when the spatial parameter of interest has a value of interest.
In some embodiments, step B.v) of the disclosed methods comprises a step of spatially separating the cells to separate the mixed cell population into subpopulations on the basis of a cell marker, such as the presence or absence or a graded level of a cell marker, for example a cell marker for cell proliferation, cell growth, fitness, cell motility, cell invasiveness, cellular metabolism, cell differentiation, DNA damage, expression levels of pre-defined genes or proteins, resistance to a compound, sensitivity to a compound, production of a compound, anoikis, senescence, apoptosis, DNA methylation or protein post-translational modification, optionally wherein the cell marker is the spatial parameter of interest.
In some embodiments, the step of spatially separating the cells is performed by FACS. In some embodiments, step B.v) of the disclosed methods further comprises a step of staining the cells with an antibody marker, such as a fluorescently labelled antibody, prior to FACS-sorting the cells, whereby cells may be spatially separated based on the signal intensity of the antibody marker.
Systems for Assessing the Effects of a Mutation of Interest in a Cell
In one aspect, the present invention provides a system comprising:
Such systems are particularly well suited for performing the methods as described herein.
In some embodiments, the synonymous mutation is a single base change compared to the target nucleic acid sequence. In some embodiments, the mutation of interest is a single base change compared to the target nucleic acid sequence.
In some embodiments, the system further comprises primers for amplifying a region comprising the target nucleic acid sequence, such as a forward and a reverse primer allowing amplification by PCR.
In some embodiments, one or more of the first oligonucleotide, the second oligonucleotide, the targeting means and the polynucleotide encoding the nuclease are comprised within a vector, such as a plasmid.
Oligonucleotides Useful for the Current Systems
Useful oligonucleotides for the disclosed systems include oligonucleotides described herein above in the section ‘Oligonucleotides useful for the current methods’.
The systems as described herein may be used for assessing the effects of several different mutations of interest in a cell at the same time. Thus, the present systems may be used for multiplex assays, wherein the effects of multiple mutations are assessed simultaneously in a single experiment.
Thus, in some embodiments, the first oligonucleotide is a plurality of first oligonucleotides each comprising a pre-determined mutation of interest. In some embodiments, the second oligonucleotide is a plurality of second oligonucleotides each comprising a pre-determined synonymous mutation.
In some embodiments, said second oligonucleotides are otherwise identical to said first oligonucleotides. In some embodiments, said second oligonucleotides are the same lengths. In some embodiments, said first oligonucleotides are the same lengths. In some embodiments, said second oligonucleotides are of different lengths. In some embodiments, said first oligonucleotides are of different lengths. In some embodiments, said second oligonucleotides are the same length as said first oligonucleotides. In some embodiments, said second oligonucleotides are of different length to said first oligonucleotides.
In some embodiments, the first oligonucleotide comprises a plurality of pre-determined mutations of interest and wherein the second oligonucleotide comprises a plurality of pre-determined synonymous mutations.
In some embodiments, the plurality of mutations is at least 2 different mutations, such as at least 3 different mutations, such as at least 4 different mutations, such as at least different mutations, such as at least 6 different mutations, such as at least 7 different mutations, such as at least 8 different mutations, such as at least 9 different mutations, such as at least 10 different mutations, such as at least 15 different mutations, such as at least 20 different mutations, such as at least 25 different mutations, such as at least different mutations, such as at least 35 different mutations, such as at least 40 different mutations, such as at least 45 different mutations, such as at least 50 different mutations or more.
In some embodiments, the target nucleic acid sequence is within a gene, a promoter or an enhancer of a gene, wherein the gene is or is suspected to be an oncogene. In some embodiments, said gene is or is suspected to be a proto-oncogene. In some embodiments, said gene is or is suspected to be a tumor suppressor gene. In some embodiments, said gene is or is suspected to be a gene encoding an enzyme such as an enzyme involved in the production of a compound such as a metabolite, a resistance gene, such as a gene involved in resistance to a compound, a pharmaceutical compound, or a pathogen such as a virus. In some embodiments, said gene is or is suspected to be a gene encoding a protein involved in cellular fitness and/or growth. In some embodiments, said gene is or is suspected to be a gene encoding a protein for any cell function, a microRNA or a long non-coding RNA. The target nucleic acid may be genomic, i.e. comprised in the genome of the cells comprised within the cell population, or it may be extrachromosomal, e.g. on a vector or plasmid or may be comprised in DNA of an infected pathogen or invading organism within another cell.
Nucleases Useful for the Current Systems
Useful oligonucleotides for the disclosed systems include oligonucleotides described herein above in the section ‘Oligonucleotides useful for the current methods’.
In some embodiments, the nuclease comprises or consist of a CRISPR/Cas nuclease and the targeting means comprise or consist of a guide RNA capable of hybridizing to the target nucleic acid sequence.
In some embodiments, the nuclease is a CRISPR/Cas nuclease, such as the Streptococcus pyogenes Cas9 nuclease of SEQ ID NO: 1, a functional variant thereof which retains nuclease activity, or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. In some embodiments, said nuclease is encoded by a polynucleotide comprising or consisting of SEQ ID NO: 6 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the nuclease is codon-optimised. For example, the nuclease is a human, codon-optimized Streptococcus pyogenes Cas9 (D10A) nickase, such as the human, codon-optimized Streptococcus pyogenes Cas9 (D10A) nickase of SEQ ID NO: 2, a functional variant thereof which retains nickase activity, or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. In some embodiments, said nuclease is encoded by a polynucleotide comprising or consisting of SEQ ID NO: 7 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the nuclease is a Francisella novicida Cas12a nuclease, such as the Francisella novicida Cas12a nuclease of SEQ ID NO: 3, a functional variant thereof which retains nuclease activity, or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. In some embodiments, said nuclease is encoded by a polynucleotide comprising or consisting of SEQ ID NO: 8 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the nuclease is a Francisella novicida Cas12a nuclease, such as MAD7 or a functional variant thereof, which retains nuclease activity.
In some embodiments, the nuclease is a Cas9-NG nuclease, such as the Cas9-NG nuclease of SEQ ID NO: 5, a functional variant thereof which retains nuclease activity, or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. In some embodiments, said nuclease is encoded by a polynucleotide comprising or consisting of SEQ ID NO: 4 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
The systems as disclosed herein may readily be used with nuclease prime editors.
In some embodiments, the nuclease is a prime editor, such as the prime editor PE2 of SEQ ID NO: 9, a functional variant thereof which retains nickase and reverse transcriptase activity, or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. In some embodiments, said nuclease is encoded by a polynucleotide comprising or consisting of SEQ ID NO: 10 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, any one of the nucleases as described herein may be codon-optimized for the cell. Thus, in some embodiments, the nuclease is codon-optimized for human cells. In some embodiments, the CRISPR/Cas nuclease, such as the Streptococcus pyogenes Cas9 nuclease of SEQ ID NO: 1 or a functional variant thereof, is codon-optimized for human cells. In some embodiments, the CRISPR/Cas nuclease, such as the Francisella novicida Cas12a nuclease of SEQ ID NO: 3 or a functional variant thereof, or such as MAD7 or a functional variant thereof, is codon-optimized for human cells. In some embodiments, the CRISPR/Cas nuclease, such as the Cas9-NG nuclease of SEQ ID NO: 5 or a functional variant thereof, is codon-optimized for human cells. In some embodiments, the CRISPR/Cas nuclease, such as the prime editor PE2 of SEQ ID NO: 9 or a functional variant thereof, is codon-optimized for human cells.
In some embodiments, the nuclease and the targeting means comprise or consist of a transcription activator-like effector nuclease (TALEN) consisting of a DNA cleavage domain and a DNA-binding domain. In some embodiments, the nuclease and the targeting means comprise or consist of a zinc finger nuclease consisting of a DNA cleavage domain and a DNA-binding domain. In some embodiments, the nuclease and the targeting means comprise or consist of a meganuclease.
Host Cells or Host Cell Populations Comprising the System for Assessing the Effects of a Mutation of Interest in a Cell
In one aspect, the present invention provides a host cell comprising the system as described herein above in the section ‘Systems for assessing the effects of a mutation of interest in a cell’.
In some embodiments, the present invention provides a host cell comprising part of the system as described herein above in the section ‘Systems for assessing the effects of a mutation of interest in a cell’.
In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is a eukaryotic cell such as a vertebrate cell, an invertebrate cell, a plant cell, a yeast cell or a fungal cell.
Uses of the Systems as Disclosed Herein for Assessing the Effects of a Mutation of Interest in a Cell
In one aspect, the present invention also provides uses of the systems as described herein above in the section ‘Systems for assessing the effects of a mutation of interest in a cell’ in a method for assessing the effects of a mutation of interest in a cell, wherein the method is as described herein above in the section ‘Methods for assessing the effects of a mutation of interest in a cell’.
In some embodiments, the cell is a mammalian cell such as a human cell. In some embodiments, the cell is a vertebrate cell. In some embodiments, the cell is an invertebrate cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a fungal cell.
In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo.
The principle of the method is illustrated in
At Day 3 after transfection, the cells were shifted to serum- and growth factor-depleted medium. (A) on Day 6, the cells were seeded on a Matrigel-coated, cell-permeable membrane in the upper chamber of a Transwell invasion chamber with serum- and growth factor-depleted medium, except that EGF was added to the lower chamber as chemo-attractant, and the cells were allowed to migrate/invade for 16 h. (B) at Day 7, the cells in the upper and lower chambers were analysed by PCR amplification of the genomic target site and NGS of the PCR product to determine the ratio of 1047R to WT* in the two cell populations. As can be seen from the results, the PIK3CA-1047R mutant was enriched in the lower chamber relative to the upper chamber, thereby demonstrating that the mutation stimulated the migrative and/or invasive properties of the cells. In conclusion, by using the method with spatial separation of cells according an initial compartment and a spatially distant compartment, the method can determine effects of a mutant on cell motile/invasive or similar properties.
Streptococcus pyogenes, (Q99ZW2)
Francisella tularensis, subspecies novicida
Streptococcus pyogenes
Francisella novicida
Items
1. A method for assessing the effects of a mutation of interest in a cell, said method comprising the steps of:
2. The method according to item 1, wherein step A.v) further comprises a step of determining an initial frequency of cells with an indel in the target nucleic acid sequence which is different from the mutation of the first and the second oligonucleotides, wherein the initial frequency of cells is determined at said initial time point, and determining a subsequent frequency of cells with an indel in the target nucleic acid sequence which is different from the mutation of the first and the second oligonucleotides, wherein the subsequent frequency of cells is determined at said subsequent time point,
3. The method according to item 2, wherein the initial and the subsequent frequency of cells with an indel is further subdivided into, respectively, an initial and a subsequent frequency of cells with an indel resulting in a frameshift mutation and an initial and a subsequent frequency of cells with an indel not resulting in a frameshift mutation, wherein a subsequent frequency of cells with an indel resulting in frameshift mutation lower or higher than the initial frequency of cells with an indel resulting in frameshift mutation indicates that the frameshift indels are affecting the cells.
4. The method according to any one of the preceding items, wherein said first oligonucleotide consists of or comprises a stretch of nucleotides identical to said second oligonucleotide except for said mutation of interest or wherein said second oligonucleotide consists of or comprises a stretch of nucleotides identical to said first oligonucleotide except for said synonymous mutation, or wherein the first oligonucleotide and the second oligonucleotide differ only in the location of the mutation of interest,
5. The method according to any one of items 1 to 3, wherein the synonymous mutation of said second oligonucleotide is located in a different position from the mutation of interest in said first oligonucleotide.
6. The method according to any one of the preceding items, wherein the parameter of interest is a temporal parameter of interest, and wherein:
7. The method according to any one of the preceding items, wherein step B.v) comprises defining at least one subpopulation of cells and a reference subpopulation of cells from the mixed population on the basis of the spatial parameter of interest and wherein:
8. The method according to any one of the preceding items, wherein step B.v) comprises a step of spatially separating the cells to separate the mixed cell population into subpopulations on the basis of a cell marker, such as the presence or absence or a graded level of a cell marker, for example a cell marker for cell proliferation, cell growth, fitness, cell motility, cell invasiveness, cellular metabolism, cell differentiation, DNA damage, expression levels of pre-defined genes or proteins, resistance to a compound, sensitivity to a compound, production of a compound, anoikis, senescence, apoptosis, DNA methylation or protein post-translational modification, optionally wherein the cell marker is the spatial parameter of interest,
9. The method according to any one of the preceding items, wherein the temporal parameter is selected from the group consisting of cell proliferation, cell growth, anchorage-independent cell growth, contribution to tumor growth, fitness, cell motility, cell invasiveness, cellular metabolism, DNA damage, expression levels of pre-defined genes and/or proteins, resistance to a compound, sensitivity to a compound, production of a compound, anoikis, senescence, contact inhibition and apoptosis.
10. The method according to any one of the preceding items, wherein the first oligonucleotide comprises at least one mutation of interest within the binding region of the nuclease, and wherein the second oligonucleotide comprises at least one synonymous mutation within the same binding region of the nuclease.
11. The method according to any one of the preceding items, wherein the first oligonucleotide is a plurality of first oligonucleotides each comprising a pre-determined mutation of interest and wherein the second oligonucleotide is a plurality of second oligonucleotides each comprising a pre-determined synonymous mutation.
12. The method according to any one of the preceding items, wherein the nuclease comprises or consists of a CRISPR/Cas nuclease and the targeting means comprise or consist of a guide RNA capable of hybridizing to the genomic target region.
13. The method according to any one of items 1 to 11, wherein the nuclease and the targeting means are selected from the group consisting of: a transcription activator-like effector nuclease (TALEN) consisting of a DNA cleavage domain and a DNA-binding domain; a zinc finger nuclease consisting of a DNA cleavage domain and a DNA-binding domain; and a meganuclease.
14. A system comprising:
15. The system according to item 14, wherein the nuclease and the targeting means are selected from the group consisting of a transcription activator-like effector nuclease (TALEN) consisting of a DNA cleavage domain and a DNA-binding domain, a zinc finger nuclease consisting of a DNA cleavage domain and a DNA-binding domain and a meganuclease.
16. A host cell comprising the system according to item 14.
17. Use of the system according to item 14 in a method for assessing the effects of a mutation of interest in a cell, preferably wherein the method is according to any one of items 1 to 13.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20211801.4 | Dec 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/084213 | 12/3/2021 | WO |
