Patent Application
20210139967
The present invention relates to a nucleic acids and variants thereof, as well as uses thereof. The present nucleic acids are useful for detecting indels (insertions and deletions) as small as 1 nucleotide in a target nucleic acid. They have a broad applicability for detecting indels following genome editing and can also be used in methods for amplifying a target nucleic acid.
The emerging gene targeting technologies for precise editing of higher eukaryote genomes such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), RNA-guided clustered regularly interspaced short palindromic repeats (CRISPRs) or Meganucleases, have revolutionized genome research and enabled studies previously limited to prokaryotes and yeast. These nuclease-based gene editing methods introduce double-stranded DNA breaks and lead to a variety of rearrangements at the breakpoint mediated by cellular repair events including non-homologous end-joining (NHEJ) and homologous recombination. In contrast to the speed by which these editing tools are being optimized and strategies for high throughput use in whole-genome screens are devised, considerably less focus is being devoted to improving capabilities for detection and characterization of the induced indels at the specific breakpoint as well as at potential off-targets. Current approaches available for identification of indels include: i) enzyme mismatch cleavage (EMC) assays, which do not provide sensitive, reliable and accurate identification of the induced indels; and ii) Sanger or next generation DNA sequencing, which is costly, time and labor intensive, and poorly suited for high throughput screening of hundreds or thousands of clones often required to select for desirable multi-allelic editing events that often occur at low frequency. Thus, methods are needed for high-throughput screening of indels.
The present invention relates to nucleic acids and variants thereof, as well as uses thereof. The present nucleic acids are useful for detecting indels (insertions and deletions) as small as 1 nucleotide in a target nucleic acid. They have a broad applicability for detecting indels following genome editing and can also be used in methods for amplifying a target nucleic acid.
(T7EI) assay of amplicons derived from a single LS174T clone (#10-8) targeted with Dual-GALNT6-ZFN. Cleaved products are indicated with asterix. Comparative IDAA of the same clone shown to the right. (b) EMC (T7EI) assay of amplicons derived from a single HeLa clone (DE4) targeted with COSMC-ZFN. Comparative IDAA of the same clone demonstrating a monoallelic −1 bp deletion (indicated with and asterix) shown to the left, relative to the intact HeLa WT peak(0). Unmarked minor light grey peaks represent the GSLIZ600 standard.
Practice of the methods, as well as preparations and use of the compositions disclosed herein employ, unless otherwise indicated, conventional techniques in molecular biology, biochemistry, bioinformatics, cell culture, recombinant DNA and related fields as are known in the art.
Adaptamer: the term herein refers to part of a nucleic acid sequence such as a primer where the adaptamer part does not hybridize to the target nucleic acid but instead is identical to the sequence of another nucleic acid such as another primer. The adaptamer is preferably comprised at the 5′-end of the primer.
Amplification reaction: an amplification reaction as understood herein is any reaction during which a target nucleic acid such as a genomic region of interest, a gene of interest, a locus of interest on a plasmid, is amplified. Polymerase chain reaction (PCR) is an example of such a reaction.
Amplicon: the term “amplicon” refers herein to a nucleic acid sequence obtained after amplification of a target nucleic acid, e.g. by PCR.
Annealing: annealing as used herein refers to the process by which complementary sequences of single-stranded nucleic acids or nucleic acid analogues pair by hydrogen bond formation, resulting in a double-stranded molecule.
Base pair (bp): a base pair shall herein refer to two complementary nucleotides linked by hydrogen bonds. The term is also used as a length measurement, interchangeably with “nt” (nucleotide).
Complementarity: as used herein, the terms “complementarity” or “complementary” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules, or there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of their hybridisation to one another.
Cutting efficiency: this term refers to the relative efficiency by which a gene targeting tool such as CRISPR/Cas9, ZFN, TALEN or other is able to induce indels at a specific locus in the genome of any given species or cell. Indels are generated after introduction of a double-strand break (DSB), i.e. induced by precise gene targeting. Subsequent to this, the double strand break is being repaired through either homologous recombination (HR) or non-homologous end joining (NHEJ). In the absence of a homologous template, non-homologous end-joining (NHEJ) is the predominant repair pathway. NHEJ repairs DSBs by joining the two ends together and usually produces no mutations, provided that the cut is clean and uncomplicated, but in some instances the repair will be imperfect, resulting in an insertion or deletion of base-pairs, producing frame-shift mutations and preventing the production of the protein of interest. The term cutting thus refers to a gene targeting tool's ability to induce double stranded breaks and indels at a predetermined site in the genome.
Denaturation: denaturation” or “melting” refers to the process by which double-stranded nucleic acid or nucleic acid analogue molecules unwind and separate into single-stranded strands through the breaking of hydrogen bonding between the bases.
Detection: the term “detection” as used herein refers to the quantitative or qualitative identification of DNA species such as, but not limited to, diffentially sized amplicons within a sample. The term “detection assay” as used herein refers to a kit, test, or method performed for the purpose of detecting an analyte nucleic acid within a sample. Detection assays produce a detectable signal or effect when performed in the presence of the target analyte, and include but are not limited to assays incorporating the processes of hybridization, nuclei acid amplification, nucleotide sequencing or primer extension. A detection assay configured for target detection is a collection of assay components that together are capable of producing a detectable signal when the target nucleic acid is present.
Downstream: as used herein, the term “downstream” applies to the end region of a nucleic acid, or to a region downstream of the nucleic acid. The term “downstream region” thus may refer to a region comprised within the target nucleic acid or the nucleic acid of interest, or to a region outside the region of interest.
Elongation primer: an elongation primer is a nucleic acid molecule capable of hybridizing or annealing to a target nucleic acid and of priming a PCR reaction.
Fluorophore/Fluorescent moiety: a fluorescent moiety or fluorophore as understood herein is any substance that can re-emit light upon excitation. Fluorescence is generated when the fluorophore, lying in its ground state, absorbs light energy at a short wavelength, creating an excited electronic singlet state, and emits light energy at a longer wavelength, creating a relaxed singlet state. The fluorophore then returns to its ground state.
Hybridisation: as used herein, the term “hybridisation” or “hybridise” is used in reference to the non-covalent, sequence-specific interaction between two complementary strands of nucleic acids into a single complex. Hybridisation and the strength of hybridisation (i.e., the strength of the association between the nucleic acids) are influenced by such factors as the degree of complementarity between the nucleic acids, the stringency of the conditions involved, the melting temperature (Tm) of the formed hybrid, and the G:C ratio within the nucleic acids.
Indel: the term “indel” stands for “insertion/deletion” and refers herein to insertion or deletion events in a nucleic acid molecule. While insertion and deletion events may occur at the same time in the same nucleic acid e.g. during gene targeting, an indel results in a net change in the number of nucleotides, typically between 1 and 50. Indels often result in frameshift mutations, except when the number of inserted/deleted nucleotides is a multiple of 3.
Melting temperature (Tm): the “Tm” or “melting temperature” or “melting point”, of an oligonucleotide refers to the temperature (in degrees Celsius) at which 50% of the molecules in a population of a single-stranded oligonucleotide are hybridised to their complementary sequence and 50% of the molecules in the population are not hybridised to their complementary sequence. The Tm can be determined empirically by means of a melting curve or it can be calculated using software well known in the art.
Nucleic acid: the term refers to a multimeric compound comprising two or more covalently bonded nucleosides or nucleoside analogues having nitrogenous heterocyclic bases, or base analogues, where the nucleosides are linked together by phosphodiester bonds or other linkages to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides, and analogues thereof, including peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Any nucleic acid analogue which will be recognised by a person skilled in the art to possess properties such that it can be used for the molecular beacons described herein can be used for embodiments of the present invention. Sugar moieties of the nucleic acid may be either ribose or deoxyribose, or similar compounds having known substitutions such as, for example, 2′-methoxy substitutions and 2′-halide substitutions (e.g., 2′-F).
Nitrogenous bases may be conventional bases (A, G, C, T, U), analogues thereof (e.g., inosine, 5-methylisocytosine, isoguanine), which include derivatives of purine or pyrimidine bases (e.g., N4-methyl deoxygaunosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases having substituent groups at the 5 or 6 position, purine bases having an altered or replacement substituent at the 2, 6 and/or 8 position, such as 2-amino-6-methylaminopurine, 06-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, and 04-alkyl-pyrimidines, and pyrazolo-compounds, such as unsubstituted or 3 -substituted pyrazolo[3,4-d]pyrimidine). Nucleic acids may include “abasic” residues in which the backbone does not include a nitrogenous base for one or more residues. A nucleic acid may comprise only conventional sugars, bases, and linkages as found in RNA and DNA, or may include conventional components and substitutions (e.g., conventional bases linked by a 2′-methoxy backbone, or a nucleic acid including a mixture of conventional bases and one or more base analogues). Nucleic acids may comprise “locked nucleic acids” (LNA), in which one or more nucleotide monomers have a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhances hybridisation affinity toward complementary sequences in single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA). Nucleic acids may comprise modified bases to alter the function or behaviour of the nucleic acid, e.g., addition of a 3′-terminal dideoxynucleotide to block additional nucleotides from being added to the nucleic acid. Synthetic methods for making nucleic acids in vitro are well known in the art although nucleic acids may be purified from natural sources using routine techniques.
Nucleotide: a “nucleotide” (or “nt”) as understood herein is a subunit of a nucleic acid consisting of a phosphate group, a 5-carbon sugar and a nitrogenous base. The 5-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is 2′-deoxyribose. The term also includes analogues of such subunits. In the present context, the unit “nt” or the unit “bp” (basepairs) will be used interchangeably to designate the length of an indel.
Polymerase Chain Reaction (PCR): the polymerase chain reaction (PCR) is a molecular biology technique allowing amplification of a single or a few copies of a nucleic acid across several orders of magnitude, generating thousands to millions of copies of a particular nucleic acid sequence. A PCR typically requires: a nucleic acid template that contains the nucleic acid region (target) to be amplified; at least two primers that are complementary to the 3′-ends of each of the sense and anti-sense strand of or surrounding the target; a polymerase; deoxynucleoside triphosphates (dNTPs; nucleotides containing triphosphate groups); a buffer solution, providing a suitable chemical environment for optimum activity and stability of the polymerase; divalent cations, magnesium or manganese ions; monovalent cation potassium ions.
Three temperatures are important and characteristic of a PCR:
i) the denaturation temperature is the temperature at which the nucleic acid present in the reaction gets denatured, i.e. shifts from being double-stranded to single-stranded. During denaturation, the sample nucleic acid becomes denatured and primers that can be hybridised to the target are released as single-stranded molecules. Thus all nucleic acids are accessible to the polymerase and complementary nucleic acid sequences can hybridise once the temperature is shifted to the annealing temperature. The denaturation temperature is typically within the range of 90° C. to 100° C. The denaturation temperature should be greater than the melting temperature of the hybridised primer-target complex. The duration of the denaturation step is determined by the user and depends on the nature of the target and of the primers used. Typically, denaturation lasts between 2 seconds and 3 minutes.
ii) the annealing temperature is the temperature at which the primers can hybridise to their complementary sequences on the target; the annealing temperature may be in the range of 43° C. to 70° C. For most applications, the annealing temperature is typically in the range of 52° C. to 65° C. The annealing temperature should be lower than the melting temperature of the hybridised primer-target complex. The duration of the annealing step is determined by the user and depends on the nature of the target and of the primers used. Typically, annealing lasts between 2 seconds and 3 minutes.
iii) the elongation temperature is the temperature at which the polymerase is active and can elongate the target; synthesis is primed when the primers are hybridised to their complementary sequence. The elongation temperature can vary depending on the nature of the polymerase. The most common elongation temperatures are 68° C. and 72° C., but any temperature in the range of 65° C. to 75° C. can be considered. The duration of the elongation step is determined by the user and depends on the nature of the target, of the polymerase and of the primers used; more particularly it depends on the length of the target molecule to be amplified and on the synthesis speed of the polymerase used.
The three cycles i), ii) and iii) are typically repeated 20 to 35 times, and may be followed by a final elongation step, often performed at the elongation temperature, to ensure full extension of any remaining single-stranded nucleic acids. In so-called 2-step PCR ii) and iii) are combined at the same temperature. The PCR can be performed in liquid phase in a thermocycler or in solid phase. For solid-phase PCR a microfluidic device may be used, in which different primer pairs can be immobilised in known positions on the surface of the device, while the fluid running through the device (also termed “reaction mixture”) contains all the other reagents necessary for the PCR, such as polymerase, template DNA, dNTPs, buffer, and primers. Alternatively, only one primer is immobilised on the device, while the other is comprised in the reaction solution.
Primer: a primer is a nucleic acid which is able to at least partly bind to a target nucleic acid. Primers are designed so that their sequence is complementary to a portion of the target nucleic acid to be amplified during an amplification reaction. In a typical PCR, two primers are used, each hybridizing on one side of the region to amplify (one primer upstream and one downstream). Primers comprise at least a sequence complementary to the target, but may also comprise additional nucleic acid sequences such as an adaptamer tail or appended extension. A primer is an oligonucleotide, which is capable of acting as a point of initiation of synthesis when placed under conditions in which production of an elongation which is complementary to a target nucleic acid strand is induced (e.g. in a PCR reaction). Primers thus allow an enzyme such as a DNA polymerase to copy the nucleic acid template (the target nucleic acid). Primers can be modified in several ways, such as labelling by e.g. a fluorophore. In order for primers to allow amplification by a polymerase, it is not necessary that the complementary sequence matches the portion of the target nucleic acid exactly; a proportion of mismatches does not prevent the reaction from occurring, if the primer is able to bind at least partly to the target sequence, as is well known in the art. Primers are sufficiently long to specifically prime the synthesis of extension products and are single stranded oligo-nucleotide sequence of usually 15-30 nucleotides or more in length and complementary in sequence to a polynucleotide target sequence, e.g. a locus contained in genomic DNA.
Target nucleic acid: the term “target nucleic acid” or “target”, when used in reference to nucleic acid detection or analysis method, refers to a nucleic acid having a particular sequence of nucleotides to be detected or analysed, e.g. in a sample suspected of containing the target nucleic acid. When used in reference to the polymerase chain reaction, “target” generally refers to the region of nucleic acid bounded by primers used in the reaction. Thus, the target is to be sorted out from other nucleic acid sequences that may be present in a sample.
Targetant: a targetant as understood herein refers to a cell or a cell population in which gene targeting events may have taken place. In the context of genome editing, a targetant is a transformant or transfectant which may be positive (successful gene targeting) or negative (unsuccessful gene targeting).
Tri-primer PCR: a tri-primer PCR is a PCR performed with three primers, where two are elongation primers typically used for PCR (a forward primer and a reverse primer each annealing to a region of the target nucleic acid to be amplified) and a universal primer. In the present context, at least one of the elongation primers comprises a tail which does not anneal to the target nucleic acid but instead functions as an adaptamer allowing a third primer to anneal to an amplicon having integrated the adaptamer sequence after at least one amplification event. The third primer can be a universal primer.
Upstream: as used herein, the term “upstream” applies to the end region of a nucleic acid, or to a region upstream of the nucleic acid. The term “upstream region” thus may refer to a region comprised within the target nucleic acid or the nucleic acid of interest, or to a region outside the region of interest.
Universal primer: a universal primer is a primer designed to anneal to a sequence complementary to the adaptamer sequence comprised within an amplicon generated using an elongation primer comprising an adaptamer. The universal primer will thus be able to hybridize to the complementary sequence of the tailed extension primer. The universal primer requires prior incorporation of the extension primer into amplicons, before its ability to prime extension and amplicon incorporation. Universal primers are sometimes labelled, e.g. by a fluorophore, either internally or externally (i.e. in the 3′- or 5′-end). Typically, when a 5′-to-3′ DNA polymerase is used, the universal primer is labelled in the 5′-end.
Wild type: The term “wild type” refers to a gene or a gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and thus is arbitrarily designed the normal or wild-type form of the gene. In contrast, the terms “modified”, “mutant” or “variant” refer to a gene or gene product that displays modifications in sequence and/or functional properties (i.e., altered abrogated function due to the introduction of indels following gene editing) when compared to the wild-type gene or gene product.
The present invention is based on the finding that the nucleic acids disclosed herein allow detection of a broad range of indels down to the single base level. The method disclosed herein is suitable for high-throughput screening based on indel detection by amplicon analysis, e.g. detection of indels induced by cellular precise gene targeting.
The present invention is as defined in the claims.
In a first aspect the invention relates to a nucleic acid comprising the sequence 5′-NNNTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 1) or a variant thereof having at least 85% identity to SEQ ID NO: 5, the 5′-end of said nucleic acid or variant thereof being labelled with a fluorophore.
In another aspect, the present invention relates to a method for detecting an indel in a target nucleic acid, said method comprising the steps of:
In yet another aspect, the invention relates to a method for synthesizing a nucleic acid as described herein.
In yet another aspect, the invention relates to the use of a nucleic acid as described herein.
In yet another aspect, the invention relates to the use of a nucleic acid as described herein in a method of detecting an indel in a target nucleic acid.
In yet another aspect, the invention relates to the use of a nucleic acid as described herein in a method of amplification of a target nucleic acid.
In yet another aspect, the invention relates to a kit comprising a nucleic acid as described herein and instructions for use.
Herein is provided a nucleic acid comprising the sequence 5′-NNNTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 1) or a variant thereof comprising a sequence having at least 85% identity to SEQ ID NO: 5, the 5′-end of said nucleic acid or variant thereof being labelled with a fluorophore. N is any nucleotide base such as a guanine base, an adenine base, a thymine base or a cytosine base. In some embodiments, the nucleic acid consists of 5′-NNNTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 1) or a variant thereof comprising a sequence having at least 85% identity to SEQ ID NO: 5. Thus the nucleic acid may comprise or consist of any of SEQ ID NO: 3 or SEQ ID NOs: 6 to 68.
In a preferred embodiment, the nucleic acid is 5′-AGCTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 3).
In some embodiments, the nucleic acid is a variant of 5′-NNNTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 1) having at least 85% identity to 5′-TGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 5). Thus in one embodiment, the variant comprises a sequence having at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as 100% identity, to SEQ ID NO: 5.
Thus in some embodiments, the nucleic acid is a variant of SEQ ID NO: 1 having between 0 and 3 mutations compared to SEQ ID NO: 5, such as 0 mutation, such as 1 mutation, such as 2 mutations, such as 3 mutations. Any nucleotide of SEQ ID NO: 5 can be mutated to any of a guanine base, an adenine base, a thymine base or a cytosine base.
The nucleic acid may further comprise an additional nucleotide base in the 5′-end. In particular, the additional base may be a guanine base, an adenine base, a thymine base or a cytosine base. In one embodiment, the additional base is a guanine base and the nucleic acid or variant thereof comprises or consists of the sequence 5′-GNNNTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 2). N is any nucleotide base such as a guanine base, an adenine base, a thymine base or a cytosine base. Thus the nucleic acid may comprise or consist of any of SEQ ID NO: 4 or SEQ ID NOs: 69 to 131.
In one embodiment, the nucleic acid comprises 5′-GAGCTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 4). In another embodiment, the nucleic acid is 5′-GAGCTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 4).
In some embodiments, the nucleic acid is a variant of 5′-GNNNTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 2) having at least 85% identity to 5′-TGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 5). Thus in one embodiment, the variant comprises a sequence having at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as 100% identity to SEQ ID NO: 5.
The nucleic acid described herein is labelled in its 5′-end with a fluorophore. Suitable fluorophores are known to the skilled person and include 6-carboxyfluorescein (6-FAM), Alexa Fluor® 350, DY-415, ATTO 425, ATTO 465, Bodipy® FL, Alexa Fluor® 488, fluorescein isothiocyanate, ATTO 488, Oregon Green® 488, Oregon Green® 514, Rhodamine Green™, 5′-Tetrachloro-Fluorescein, ATTO 520, 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluoresceine, Yakima Yellow™ dyes, Bodipy® 530/550, hexachloro-fluorescein, Alexa Fluor® 555, DY-549, Bodipy® TMR-X, cyanine phosphoramidites (cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5), ATTO 550, TAMRA (carboxy-tetramethyl-rhodamine), Rhodamine Red™, ATTO 565, Carboxy-X-Rhodamine, Texas Red (Sulforhodamine 101 acid chloride), LightCycler® Red 610, ATTO 594, DY-480-XL, DY-610, ATTO 610, LightCycler® Red 640, Bodipy 630/650, ATTO 633, Alexa Fluor® 647, Bodipy 650/665, ATTO 647N, DY-649, LightCycler® Red 670, ATTO 680, LightCycler® Red 705, DY-682, ATTO 700, ATTO 740, DY-782, IRD 700 and IRD 800, CAL Fluor® Gold 540 nm, CAL Fluor® Gold 522 nm, CAL Fluor® Gold 544 nm , CAL Fluor® Orange 560 nm, CAL Fluor® Orange 538 nm, CAL Fluor® Orange 559 nm, CAL Fluor® Red 590 nm, CAL Fluor® Red 569 nm, CAL Fluor® Red 591 nm, CAL Fluor® Red 610 nm, CAL Fluor® Red 590 nm, CAL Fluor® Red 610 nm, CAL Fluor® Red 635 nm, Quasar® 570 nm, Quasar® 548 nm, Quasar® 566 nm (Cy 3), Quasar® 670 nm, Quasar® 647 nm, Quasar® 670 nm (Cy 5), Quasar® 705 nm, Quasar® 690 nm, Quasar® 705 nm (Cy 5.5), Pulsar® 650 Dyes, SuperRox® Dyes.
In some embodiments, the fluorophore is 6-carboxyfluorescein (6-FAM).
Thus in some embodiments, the nucleic acid is as described above and is labelled in its 5′-end by a fluorophore, such as 6-FAM. The following nucleic acids are thus provided: 6-FAM-AGCTGACCGGCAGCAAAATTG-3′, 6-FAM-GAGCTGACCGGCAGCAAAATTG-3′. Any of SEQ ID NOs: 3, SEQ ID NO: 4 or SEQ ID NOs: 6-131 may be labelled with a fluorophore in their 5′-end; in particular embodiments, the fluorophore is 6-FAM.
It will be understood that the fluorophore should be selected such that it is suitable for labelling the 5′-end of a nucleic acid described herein. The 3′-end and the 5′-end of nucleic acid molecules differ in that the 5′-end usually bears a free 5′-phohsphate group, while the 3′-end bears a free 3′-hydroxyl group. Some fluorophores are able to bind only one of the ends (either 3′ or 5′), while others are capable of binding both ends. The fluorophores suitable for labelling the nucleic acids described herein are preferably capable of labelling the 5′-end exclusively, or they are capable of labelling the 5′-end and the 3′-end, provided that labelling in the 3′-end does not prevent the labelled nucleic acid from acting as a primer in a reaction for amplifying a target nucleic acid.
Also provided herein is a use of the nucleic acids of the invention.
The nucleic acids disclosed herein can be used in a method for detecting indels by amplicon analysis. The nucleic acids can also be used in a method for amplification of a target nucleic acid.
Thus there is also provided herein a method for detecting an indel in a target nucleic acid, said method comprising the steps of:
The inventors have found that the nucleic acids disclosed herein are particularly useful for detecting indels. Indels are, as defined above, insertion or deletion events which can occur for example during gene targeting or gene editing processes, and which result in a net change in the number of nucleotides in a nucleic acid. One of the bottlenecks of many gene editing methods routinely used is the identification of targetants carrying indels. The nucleic acids described herein can be used in a method for identifying indels in a target nucleic acid with a sensitivity of a single nucleotide. The method is based on a triprimer amplification reaction, such as a triprimer PCR. One of the primers is a first elongation primer which is capable to hybridize or anneal to an upstream or downstream region of a target nucleic acid to be amplified. A second elongation primer is capable to hybridize or anneal to another region of the target nucleic acid to be amplified. If the first elongation primer hybridizes to an upstream region, the second elongation primer hybridizes to a downstream region, and vice versa, so that the set of elongation primers can be used in a normal PCR reaction to amplify the target nucleic acid. At least one of the elongation primers comprises an adaptamer sequence, while at most one of the elongation primers hybridizes or anneals to the target nucleic acid sequence substantially over its whole length. At least one other primer is provided which is a universal primer. In some embodiments, the universal primer is a nucleic acid as defined above, and is identical to the adaptamer of at least one of the elongation primers. Enzymes, nucleotides, and other reagents required for amplifying the target nucleic acid are also provided. The amplification reaction is then performed. The end products of the reaction are amplicons which are labelled by the fluorophore. The amplicons are then analysed by suitable methods, such as DNA capillary electrophoresis. The principle of the method is outlined in
Although the above method is illustrated in
It will also be understood that the method can be adapted so that both elongation primers have an adaptamer sequence, which may be identical or different. In some embodiments, the first elongation primer comprises a first adaptamer which has a sequence identical to a first universal primer and the second elongation primer comprises a second adaptamer which has a sequence identical to a second universal primer. The first and second universal primers may differ in sequence. The first and second universal primers may have any of SEQ ID NOs: 3, 4, or SEQ ID NOs: 6-131 as detailed above. The first and second universal primers may be labelled with the same or with different fluorophores.
The method can also be carried out with the first and the second elongation primers comprising the same adaptamer sequence which can be recognised by at least one universal primer. The universal primers recognising the adaptamer sequence may have identical or similar sequences. If the sequences are identical, the universal primers recognising the adaptamer can be labelled with the same fluorophore or with different fluorophores.
The sequence identity between the adaptamer and the universal primer is sufficient to allow hybridization of the universal primer to an amplicon generated with elongation primers where at least one elongation primer comprises the adaptamer. Thus in some embodiments, the sequence identity between the adaptamer and the universal primer is 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%, such as 100%.
The desirable length of the region of the elongation primers that is capable of hybridizing to the target nucleic acid can be any length suitable for amplifying the target nucleic acid. Thus in some embodiments, the length of the first and/or of the second elongation primers is between 5 and 100 nucleotides, such as between 6 and 90 nucleotides, such as between 7 and 80 nucleotides, such as between 8 and 70 nucleotides, such as between 9 and 60 nucleotides, such as between 10 and 50 nucleotides, such as between 11 and 45 nucleotides, such as between 12 and 40 nucleotides, such as between 13 and 35 nucleotides, such as between 14 and 30 nucleotides, such as between 15 and 25 nucleotides, such as between 16 and 24 nucleotides, such as between 17 and 23 nucleotides, such as between 18 and 22 nucleotides, such as between 19 and 21 nucleotides, such as 20 nucleotides. The optimal length of the hybridizing region of the elongation primers may depend on the sequence of the primers, on the sequence of the target nucleic acid, such as for example their GC content. The skilled person knows how to design suitable elongation primers.
In a preferred embodiment, the at least three primers (two elongation primers and at least one universal primer) are provided simultaneously and the reaction, e.g. the PCR, is performed with all three primers present at the same time as the reaction progresses. In another embodiment, the two elongation primers are provided in a first part of the reaction; this results in a first set of amplicons comprising the adaptamer sequence. The universal primer can thus be provided in a subsequent stage, optionally with additional amounts of at least the elongation primer which hybridizes in the end opposite to the end comprising the adaptamer sequence, and the first set of amplicons resulting from the first step are amplified and labelled in this second step.
The amplification reaction may be any reaction known in the art allowing amplification of a target nucleic acid. Such methods are known in the art, and include PCR, qPCR, RT-PCR, and variations thereof, such as allele-specific PCR, Assembly PCR, Asymmetric PCR, Dial-out PCR, Digital PCR, Helicase-dependent amplification, hot start PCR, intersequence-specific PCR, inverse PCR, ligation-mediated PCR, methylation-specific PCR, multiplex ligation-dependent probe PCR, multiplex PCR, nanoparticle-assisted PCR, nested PCR, overlap-extension PCR, solid phase PCR, suicide PCR, thermal asymmetric interlaced PCR, touchdown PCR and universal fast walking.
The method of the present invention may comprise the step of adding other reagents necessary for performing such amplification reactions. In the case of PCR reaction, such reagents include nucleotides (A, T, G, C), a DNA polymerase, buffer, optionally salts such as magnesium salts, e.g. MgCl2. Routine optimisation may be required in order to determine the optimal temperature at which the reaction is most efficient and/or specific. PCR reactions are performed in thermal cyclers known in the art.
The invention thus relates to a method of detecting indels, where the universal primer is a nucleic acid as described herein. In some embodiments, the universal primer comprises or consists of the sequence 5′-NNNTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 1) or a variant thereof comprising a sequence having at least 85% identity to SEQ ID NO: 5, the 5′-end of said universal primer being labelled with a fluorophore. In some embodiments, the universal primer consists of 5′-NNNTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 1) or a variant thereof comprising a sequence having at least 85% identity to SEQ ID NO: 5. Thus the universal primer may comprise or consist of any of SEQ ID NO: 3 or SEQ ID NOs: 6 to 68.
In a specific embodiment, the universal primer has the sequence 5′-AGCTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 3) and is labelled at its 5′-end by a fluorophore. In a particular embodiment, the universal primer is SEQ ID NO: 3 labelled with 6-FAM.
In some embodiments, the universal primer is a variant of 5′-NNNTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 1) having at least 85% identity to 5′-TGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 5). Thus in one embodiment, the variant comprises a sequence having at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as 100% identity, to SEQ ID NO: 5.
The universal primer may further comprise an additional nucleotide base in the 5′-end. In particular, the additional base may be a guanine base, an adenine base, a thymine base or a cytosine base. In one embodiment, the additional base is a guanine base and the nucleic acid or variant thereof comprises or consists of the sequence 5′-GNNNTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 2). N is any nucleotide base such as a guanine base, an adenine base, a thymine base or a cytosine base. Thus the nucleic acid may comprise or consist of any of SEQ ID NO: 4 or SEQ ID NOs: 69 to 131.
In a specific embodiment, the universal primer comprises or consists of 5′-GAGCTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 4). In a particular embodiment, the universal primer is 5′-GAGCTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 4) and is labelled at its 5′-end by a fluorophore. In a particular embodiment, the universal primer is SEQ ID NO: 4 labelled with 6-FAM.
In some embodiments, the universal primer is a variant of 5′-GNNNTGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 2) having at least 85% identity to 5′-TGACCGGCAGCAAAATTG-3′ (SEQ ID NO: 5). Thus in one embodiment, the variant comprises a sequence having at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity, such as 100% identity to SEQ ID NO: 5.
The adaptamer comprised within at least one of the elongation primers is identical to the universal primer. Mismatches are possible to the extent that they do not prevent annealing of the universal primer to the adaptamer. Thus in some embodiments, the sequence identity between the adaptamer and the universal primer is 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%, such as 100%.
The universal primer preferably is not capable of binding or annealing or hybridizing to any sequence of the target nucleic acid or of the DNA material in which the target nucleic acid is comprised or of any DNA material present in the reaction. For example, if the target nucleic acid has been isolated from a cell together with other DNA material, the DNA material and the target nucleic acid do not comprise a sequence identical, complementary to or substantially identical or complementary to the sequence of the universal primer. Without being bound by theory, it is expected that such binding would reduce the efficiency, sensitivity and/or specificity of the present method.
As shown in Example 6, no exact match was found in any of the species of which the genomes are available at NCBI per November 2014 for the nucleic acids having SEQ ID NO: 3 or SEQ ID NOs: 6-68 (see Example 6). Without being bound by theory, it is expected that this is one of the reasons why the present method displays such high specificity.
Routine optimisation may be needed in order to determine the optimal ratio between the first elongation primer, the second elongation primer and the universal primer. By optimal ratio is understood the relative amounts of each primer resulting in optimal reaction specificity and in optimal yield. In some embodiments, the ratio (universal primer):(first elongation primer):(second elongation primer), where the first elongation primer is the primer comprising the adaptamer, is between 1:1:1 and 20:1:20, such as 2:1:2, such as 3:1:3, such as 4:1:4, such as 5:1:5, such as 6:1:6, such as 7:1:7, such as 8:1:8, such as 9:1:9, such as 10:1:10, such as 11:1:11, such as 12:1:12, such as 13:1:13, such as 14:1:14, such as 15:1:15, such as 16:1:16, such as 17:1:17, such as 18:1:18, such as 19:1:19, such as 2:1:3, such as 3:1:4, such as 4:1:5, such as 5:1:6, such as 6:1:7, such as 7:1:8, such as 8:1:9, such as 9:1:10, such as 10:1:11, such as 11:1:12, such as 12:1:13, such as 13:1:14, such as 14:1:15, such as 15:1:16, such as 16:1:17, such as 17:1:18, such as 18:1:19, such as 19:1:20, such as 3:1:2, such as 4:1:3, such as 5:1:4, such as 6:1:5, such as 7:1:6, such as 8:1:7, such as 9:1:8, such as 10:1:9, such as 11:1:10, such as 12:1:11, such as 13:1:12, such as 14:1:13, such as 15:1:14, such as 16:1:15, such as 17:1:16, such as 18:1:17, such as 19:1:18, such as 20:1:19. As the skilled person knows, the optimal primer ratio is dependent on many parameters, including the exact sequence of the elongation primers, which influences the hybridization temperature and the possible formation of secondary structures which might interfere with the reaction efficiency. Another parameter may be the nature of the target nucleic acid: some target regions are more easily accessible to primers than others depending of their environment or on the secondary structures they may adopt. Without being bound by theory, the inventors have found that it is often advantageous that the primer which comprises the adaptamer sequence be present in a smaller amount than the other elongation primer and the universal primer, which themselves are preferably present in equimolar amounts. Such ratios have been observed to increase specificity of the reaction.
Other parameters such as the number of cycles or the temperatures of the different steps of the amplification reaction may also need routine optimisation. The skilled person knows how to optimise such parameters.
After the amplification reaction has been performed, the size of the fluorophore-labelled amplicons thus obtained is analysed.
Methods for analysing the size of the labelled amplicons are available to the skilled person. For example, the fluorophore-labelled amplicons are detected by DNA capillary electrophoresis (CE), melting curve analysis, polyacrylamide gel electrophoresis or other size exclusion methods where laser induced fluorophore detection can be applied. Suitable equipment for analysing amplicon size is known to the skilled person and includes e.g. genetic analysers.
The method described herein can detect insertions or deletions that are very small, i.e. down to a single nucleotide insertion or deletion. Thus in one embodiment, the method allows detection of an indel of at the most 10 nucleotides, such as 9 nucleotides, such as 8 nucleotides, such as 7 nucleotides, such as 6 nucleotides, such as 5 nucleotides, such as 4 nucleotides, such as 3 nucleotides, such as 2 nucleotides, such as 1 nucleotide at the most.
The method of the present invention is thus particularly well suited for high-throughput analysis of targetants following gene editing or gene targeting. Methods of gene editing or gene targeting are known in the art and include, but are not limited to: zinc-finger endonuclease (ZFN) editing; TALEN-mediated editing; CRISPR-Cas-based methods; targeted mutagenesis using primers comprising the desired mutations; random mutagenesis;
integrative plasmids. Such gene editing or targeting methods sometimes result in off-target effects and/or creation of indels and/or other undesirable effects, as explained above.
The target nucleic acids to be analysed using the present method may be of several kinds. In some embodiments, the target nucleic acid is comprised or has been comprised within a cell, for example in a genome of a cell, or on a plasmid. In some embodiments, the target nucleic acid is a viral nucleic acid.
In particular embodiments, the cell is a eukaryotic cell. The eukaryotic cell may be selected from the group consisting of a human cell, a Chinese hamster cell, a murine cell, a rat cell, an insect cell such as an Sf9 cell, a canine cell, a plant cell, an old world monkey cell, a new world monkey cell, a pig cell, a horse cell, a bovine cell, a goat cell, a lamb cell, a fish cell, an avian cell, a feline cell and a yeast cell such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Pichia pastoris.
In other embodiments, the eukaryotic cell is a plant cell. The plant cell may be from a genus such as the family Brassicaceae such as Arabidopsis thaliana, the Solanaceae, or nightshade genus such as tomato, Solanum tuberosumor potato, cereal grain such as rice or wheat, corn or maize or other.
In other embodiments, the cell is a prokaryotic cell. The prokaryotic cell may be a bacterial cell. The cell may originate from a bacteria selected from the group of Escherichia sp. such as E. coli, Lactobacillus sp., Streptomyces sp. Campylobacter sp., Salmonella sp., Listeria sp., Staphylococcus sp. Bacillus sp., and Clostridium sp.
In some embodiments, isolation of the DNA material of the cell comprising the target nucleic acid is required. Methods for isolating DNA from cells are known in the art. In other embodiments, the amplification reaction is performed directly on cell samples without prior DNA extraction. In such embodiments, cells may be lysed prior to amplification, as is known to the skilled person. Lysis can be performed by incubating the cells at high temperatures, or by using lysing agents known in the art. Whether DNA extraction is necessary will depend on factors such as e.g. the nature of the cell and the nature of the target nucleic acid.
In specific embodiments, the cell comprising the target nucleic acid to be analysed with the present method is comprised or has been comprised within a pool of cells, for example a pool of targetants. The targetants within the pool may be genetically identical (i.e. propagated from a single cell or clone) or different.
In some embodiments, the present method is suitable for high-throughput screening of targetants.
Thus the present method is useful for screening targetants obtained after gene or genome editing. The targetants may be the result of strategies involved methods of genome editing known in the art. In some embodiments, the targetants are obtained after cloning using transfection with DNA fragments having homology to a target region. In other embodiments, the targetants are obtained after random mutagenesis. In yet other embodiments, integrative plasmids are used. In yet other embodiments, the genome editing is performed using ZFNs, TALENS or CRISPR systems. It is to be understood that the present method is not limited to targetants obtained by particular gene editing strategies.
The present method is also useful for determining gene targeting efficiency.
The present invention also relates to a nucleic acid as defined above for use in a method of detecting an indel in a target nucleic acid.
In some embodiments, the indel is an insertion and/or a deletion resulting in a net change of the total number of nucleotides within a nucleic acid, where the net change is equal to m, where m is an integer ≥1. Thus in specific embodiments, the desired size of the target nucleic acid (unmodified) is S and the indel is an insertion, so that the size of the nucleic acid amplified with the present method is S+m, wherein m is at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10. In other specific embodiments, the indel is a deletion and the size of the nucleic acid amplified with the present method is S−m, wherein m is at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10.
Accordingly, in some embodiments, the nucleic acids of the present invention can be used in a method of detecting an indel in a target nucleic acid, where the indel is an insertion or a deletion of 1 nucleotide or more, such as at least 2 nucleotides, such as at least 3 nucleotides, such as at least 4 nucleotides, such as at least 5 nucleotides, such as at least 6 nucleotides, such as at least 7 nucleotides, such as at least 8 nucleotides, such as at least 9 nucleotides, such as at least 10 nucleotides, or more.
Detecting indels in the resulting products with the method described herein has numerous potential applications, such as, but not limited to, diagnosing a disease or a disorder. As an example, the nucleic acids disclosed herein can be used for detecting microsatellite expansion, contraction, trinucleotide repeat disorders. Trinucleotide repeat disorders are typically due to trinucleotide repeat expansion events, and comprise polytglutamine diseases, wherein the glutamine-encoding codon CAG is repeated, and non-polyglutamine diseases, which involve repeats of other trinucleotides.
Examples of diseases or disorders resulting from such mechanisms are Dentatorubropallidoluysian atrophy, Huntington's Disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia type 1, 2, 3, 6, 7, 8, 12 or 17, fragile X syndrome, Fragile X-associated tremor/ataxia syndrome, fragile XE mental retardation, Friedreich's ataxia and myotonic dystrophy.
In one aspect, the invention relates to a method for synthesizing a nucleic acid of the invention.
Methods for synthesizing nucleic acids as disclosed herein are known to the skilled person. Such methods include, but are not limited to, chemical oligonucleotide synthesis methods such as solid-phase synthesis. The synthetic oligonucleotides can then be released from the solid phase to solution and collected.
Methods for fluorescently labelling the synthetic oligonucleotides are also known to the skilled person. In the present context, fluorescent labelling is the process of covalently attaching a fluorophore to a nucleic acid. This is typically accomplished using a reactive derivative of the fluorophore that selectively binds to a functional group contained in the target molecule. Common reactive groups include, but are not limited to: isothiocyanate derivatives such as FITC and TRITC (derivatives of fluorescein and rhodamine), succinimidyl esters such as NHS-fluorescein, maleimide activated fluorophores such as fluorescein-5-maleimide, or phosphoramidite reagents containing protected fluorescein and other fluorophores, e.g. 6-FAM phosphoramidite 2. Phosphoramidte agents are can be reacted with hydroxy groups to allow the preparation of fluorophore-labelled oligonucleotides.
Also provided herein is a kit comprising a nucleic acid as defined herein and instructions for use. The kit may further comprise reagents required for performing an amplification reaction and/or detecting indels and/or for performing the method of the invention.
Precise Gene Targeting Induced Indel Detection by Amplicon Analysis (IDAA)
All primers used were obtained from TAGC Copenhagen A/S, Denmark. Amplicons were fluorophore labeled by tri-primer amplification using a universal 6-FAM 5′-labelled primer FamF and primers flanking the gene editing target site of which the sense primer carried a FamF target sequence extension. For each of the following genes, the universal primer had the sequence AGCTGACCGGCAGCAAAATTG (SEQ ID NO: 3): hCOSMC, mCosmc, cCosms, hGALNT6, cSt6galnac2, hKRAS,
Optimal tri-primer generated amplicon yields were observed using a PCR primer ratio of 10:1:10 (FamF:XF:XR), X being either hCOSMC, hGALNT6, hKRAS, mCosmc, cSt6galnac2 or cCosmc. PCR was performed in 25 μl, using AmpliTaq Gold (ABI/Life Technologies, USA) or TEMPase Hot Start DNA Polymerase (Amplicon, Denmark), 0.5 μM:0.05 μM:0.5 μM (FamF:F:R) primers and a touchdown thermocycling profile using an initial 72° C. annealing temperature ramping down by 1 degree/cycle to 58° C., followed by an additional 25 cycles using 58° C. annealing temperature. Denaturing and elongation were performed at 95° C. for 45 sec and 72° C. for 30 sec respectively. 1 μl of the PCR reaction or dilutions thereof was mixed with 0.5 μl LIZ600 or LIZ500 size standard (ABI/Life Technologies, USA) and applied to fragment analysis on ABI3010 sequenator (ABI/Life Technologies, USA) using conditions recommended by the manufacturer. Raw data obtained was analysed using Peak Scanner Software V1.0 (ABI/Life Technologies, USA).
CompoZr® ZFN plasmids for human C1GALT1C1/COSMC, mouse C1galt1c1/Cosmc and human GALNT6 were obtained from Sigma (Sigma-Aldrich, St. Louis, Mo., USA). GeneArt® TALEN St6galnac2 CHO plasmids were obtained LifeTechnologies (Thermo Fisher Scientific Inc, Waltham, Mass., USA). Cas9 plasmid was codon optimized for CHO expression. Four CHO Cosmc gRNA targets were selected using a tool developed for Cas9/gRNA target prediction (staffbiosustain.dtu.dk/laeb/crispy/). Dual GALNT6 expression vector was constructed as follows: GFP and the two GALNT6 ZFN1/2 sequences were fused via 2A peptide as outlined in
HeLa, DE4, HEK293AC2, mouse MC57 cells were cultured in DMEM with 10% FBS and 1% L-glutamine, and K562 cells were cultured in Iscove's modified Dulbecco's medium, 10% FBS and 1% L-glutamine. CHO cells were cultured in ex-Cell-CD media (Sigma Aldrich, USA) with 2% L-glutamine. Cells were nucleofected using solution kits T and V (K562) (Lonza, USA) and a Amaxa® Cell Line Nucleofector® device as previously described using protocols provided by Lonza. In brief, 1×106 cells were transfected with 2 μg of ZFN or TALEN plasmid pairs or 2 μg of Dual-GALNT6-ZFN. For CRISPR/Cas9 CHO Cosmc targeting, 2 μg Cas9 and gRNA plasmids were nucleofected, and for pCMV-Cas9-GFP expressing KRAS gRNA 2 μg plasmid was used. Cells were exposed to a cold shock 30° C. for 2 days post-transfection, and incubated one day at 37° C. after which DNA of the cell pool was prepared using Nucleospin kit as recommended by the supplier (Machery-Nagel, USA).
For consecutive targeting of the KRAS locus, K562 cells were subjected to FACS 3 days after nucleofection for isolation of the 2% most highly GFP fluorescent cells that were then cultured for about 1 week. Thereafter, an aliquot of the cell pool was analysed by IDAA (1st hit), whereas the rest of the cells were subjected to another two rounds of nucleofection and FACS to produce 2nd and 3rd hit pools, respectively, Furthermore, after the 3rd hit, cells were also single-cell plated in 96-well plates and expanded to clonal cell lines.
Expanded single cell clones were lysed in the wells using QuickExtract DNA extraction solution (Sigma-Aldrich, USA), 1 μl lysate was used for target region amplification. Amplified products were band purified using Qia-mini elute purification (Qiagen Inc, USA) and used for Topo-ligation into pCR4-Topo vector (Invitrogen/Life Technologies, USA), transformed into MegaXcells (Invitrogen/Life Technologies, USA) and LB-Streptomycin/Carabenicillin plated. A custom based direct sequencing protocol was developed by which large sized single cell colonies were boiled in 10 μl TE for 10 min and 5 μl hereof added to BigDye.3.1 reaction (ABI/Life Technologies, USA) and sequenced using 45× sequencing cycles (BGI Europe, Denmark).
Endonucleolytic heteroduplex DNA cleavage analysis was performed using T7-nuclease-I (New England Biolabs, USA) as recommended by the supplier. In brief, heteroduplex and/or perfect match amplicons were incubated with 1 μl T7nuclease in a 20 μl volume at 37° C. for 1 h, followed by 3% agarose gel GelStar (Lonza, USA) analyses.
K562 cells were nucleofected as described above. After 1, 2 or 5 days, aliquots of the cells were harvested and lysed in SDS-PAGE sample buffer. The cell lysates were normalized for protein content and equal amounts of protein were subjected to immunoblotting. Blots were incubated with primary antibodies to the proteins indicated (beta-Actin: Abcam, cat. number: ab8226; c-Myc: Santa Cruz Biotechnology, cat. number: sc-40; Flag: Sigma-Aldrich, cat. number: F3165) followed by HRP-conjugated secondary antibodies (Dako, Denmark), both for 1 hr at room temperature and finally developed with ECL (Pierce/Thermo Scientific, USA). For immunocytochemistry (ICC), CHO cells were fixed and stained on Teflon coated slides. In brief, cells were dried on slides and incubated overnight, 4° C., with the monoclonal antibody 5F4, followed by secondary anti-mouse-Ig-FITC incubation (Dako, Denmark), visualization and imaging by fluorescence microscopy.
We have developed a single-step tri-primer PCR setup with a universal 6-FAM 5′-labelled primer (FamF), which is designed to be specific for an extension placed on one of the target specific primers, and thus enabling one-step fluorophore labelling of amplicons derived from any given target using a universal single set-up condition (
To demonstrate the applicability of the IDAA strategy in nuclease-based genome editing, we first demonstrated its use for evaluating cutting efficiencies of CRISPR/Cas9 targeting using four different gRNA designs (
IDAA analysis of the four cell pools obtained after CRISPR/Cas) gRNA 2 days post transfection shows that the total cutting efficiencies for gRNA1 and gRNA2 are 23% and 46%, respectively, see
We found consistent targeting efficiencies of total cell pools when using either IDAA or phenotypic screening 3d after transfection with the constructs (
Taken together the results demonstrate the usefulness of IDAA in determining cutting efficiencies of gene targeting tools such as CRISPR/Cas9, ZFN, TALEN or other in cell pools and single cell clones, down to +/−1 bp differences. The example in particular demonstrates the usefulness of IDAA in determining the efficacies of gRNA designs with unknown CRISPR/Cas9 cutting efficacy and as such IDAA is ideally suited for validating the efficiency of a multitude of gRNA designs.
We next demonstrate that IDAA is amenable for high throughput screening of targeted individual cell clones with a discrimination power down to a single base indel. We used the same CRISPR/Cas9 Cosmc gRNA2 targeted pool for single cell cloning shown in
To corroborate the usability of IDAA for multi-allele gene targeting, bi-allelic CHO St6galnac2 and tri-allelic human K562 KRAS were targeted with TALEN and CRISPR/Cas9 nucleases respectively. The IDAA results for TALEN CHO St6galnac2 two days after transfection clearly detected indels in the cell pool and successful bi-allelic targeting was obtained in a substantial fraction (48%) of FACS single cell clones analysed (
We further showed that the IDAA strategy is ideal for ZFNs, which generally have lower cutting efficiencies. We first tested a ZFN with medium cutting efficiency (18% evaluated by an EMC assay, Sigma-Aldrich) targeting the Cosmc gene in a murine cell line (MC57) (
These data show that IDAA can successfully be used to detect indels as small as 1 bp, even when such events occur at low frequencies, while they are not detected by the EMC assay.
We demonstrate that IDAA is ideally suited for fast and simple screening for indels in candidate off-target genes identified by target sequence similarity, which is a major concern for all precise gene editing strategies. We first screened the CHO genome for the most likely off-target sites for gRNA2 targeting Cosmc, and identified a total of 189 potential off-targets with two to four bp mismatches (
This example shows that IDAA can be used to demonstrate the absence of off-target events in clones to be tested.
The present IDAA method is ideally suited for the detection of stable, heritable precise gene edited indels in target cells to be used for therapeutic purposes.
This example demonstrates IDAA's use in investigation and identification of ZFN-mediated indels at the CXCR4 locus in primary T cells. Fresh CD4+ T cells from live human donors are obtained from Blodbanken, Alborg Sygehus Nord, Denmark. 2.5×106 CD4+ cells are seeded at a density of 0.8 ×106 in RPMI containing 10% fetal calf serum, 1% penicillin/streptomycin, and 100U/ml interleukin-2. 1×106 cells are nucleofected with 2 μg of each of the CXCR4 ZFN (CompoZr® Knockout ZFN Kit, Sigma-Aldrich, USA) using Amaxa nucleofector (Lonza, USA) using protocols and reagents as described by the manufacturer. Cells are treated essentially as explained in example 1. Two days post transfection the CXCR4 treated cell pool is examined by IDAA for quantification of targeting efficiency as decribed in example 2. Tri-primer CXCR4 target specific primers; CXCR4 extension primer (5′-GAGCTGACCGGCAGCAAAATTGCAACCTCTACAGCAGTGTCCTCATC-3′ (SEQ ID NO: 133) adaptamer sequence underlined)/CXCR4REV (5′-GGAGTGTGACAGCTTGGAGATG-3′ and universal primer (SEQ ID NO: 3) were used to fluorophore label CXCR4 amplicons for IDAA as described in example 1. Mutation frequencies obtained by IDAA match the described frequencies obtained by CXCR4 ZFN manufacturer (Sigma-Aldrich). Cells abrogated for CXCR4 expression due to CXCR4 targeting will be resistant to natural CXCR4-tropic HIV strains as described in (CA Didigu et al., 2014, Blood, 123, p 61-69) and as such, the percent of CXCR4 gene disrupted population increases after CXCR4-tropic HIV infection due abrogated CXCR4 coreceptor expression blocking the ability of X4-tropic virus to infect cells and conferring a survival advantage to the cell population modified by the CXCR4-specific ZFNs. Such HIV-resistant CD+ T cells are to be considered for therapeutic treatment of HIV infected individuals.
This example demonstrates that the present method and nucleic acids can be used for therapeutic purposes.
In conclusion, the IDAA strategy presented here enables sensitive, precise and reliable identification of indels in a high throughput mode providing detailed information of cutting efficiency, size and nature of allelic variants generated by any of the precise gene editing technologies. The IDAA strategy is user friendly and easily implemented in any standard laboratory and can greatly advance implementation and use of precise gene targeting.
SEQ ID NO: 4 and variants thereof (SEQ ID NOs: 134-187, SEQ ID NOs: 71, 75, 79, 81-83, 98, 114, 130) were analysed as follows. All positions except for the first G-nucleotide were substituted for any of the 4 bases (A, T, G and C) and all individual permutations were BLAST analysed against the total species NCBI database as of November 2014. No exact matches were found in any of the species.
Mismatch hits and unique number of taxa hits (taxa being any group or rank in a biological classification into which related organisms are classified) were analysed in human and mouse; mismatches were allowed at any position and SEQ ID NO: 4 was used as query. The results are shown in Table 1:
As can be seen for queries with only one mismatch, no identical sequences were found.
When allowing 1 to 4 mismatches, 917 unique taxa hits were retrieved (data not shown).
SEQ ID NO: 3 and variants thereof (SEQ ID NOs: 188-241 and SEQ ID NOs: 8, 12, 16, 18-20, 35, 51, 67) were analysed as follows. All positions were substituted for any of the 4 bases (A, T, G and C) and all individual permutations were BLAST analysed against the total species NCBI database as of November 2014. No exact or 1 bp matches were found in any of the species.
Mismatch hits and unique number of taxa hits (taxa being any group or rank in a biological classification into which related organisms are classified) were analysed in human and mouse; mismatches were allowed at any position and SEQ ID NO: 3 was used as query. The results are shown in tTable 2:
As can be seen for queries with one or two mismatches, no identical sequences were found.
When allowing 2 to 4 mismatches, 472 unique taxa hits were retrieved (data not shown).
Steentoft, C. et al. Nat. Methods 8, 977-82 (2011)
Duda, K. et al. Nucleic Acids Res. 1-16. April 21. [Epub ahead of print] (2014)
Steentoft, C. et al. EMBO J. 32, 1478-88 (2013)
| Number | Date | Country | Kind |
|---|---|---|---|
| PA201470809 | Dec 2014 | DK | national |
This application claims priority to U.S. patent application Ser. No. 15/536,440, filed Jun. 15, 2017, which is a U.S. national stage application of PCT/DK2015/050405, filed Dec. 18, 2015, whih claims priority to Danish application No. PA201470809, filed Dec. 19, 2014. The entire content of each application is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15536440 | Jun 2017 | US |
| Child | 17113452 | US |
