CN116656784B - Titer measurement method of amplification-free adeno-associated virus genome - Google Patents
Titer measurement method of amplification-free adeno-associated virus genome Download PDFInfo
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Abstract
The application provides an amplification-free adeno-associated virus (AAV) titer determination method, corresponding crRNA, fluorescent DNA probes and a kit, and particularly relates to a method for detecting adeno-associated virus (AAV) titer based on a CRISPR-Cas12a system. The determination method provided by the application not only does not need PCR amplification, but also has low requirements on a test instrument, and can simply, rapidly, accurately and stably determine the titer of the adeno-associated virus genome.
Description
Technical Field
The application relates to the field of biotechnology, in particular to a titer measurement method of an amplification-free adeno-associated virus genome, and corresponding crRNA, single-stranded DNA probes and kits.
Background
Adeno-associated virus (AAV) vectors are DNA containing a single strand, non-enveloped, belonging to the helper-dependent parvovirus B19 family. This virus was found in Atchison et al 1965 in Simian adenovirus 15 (SV-15) and was defined as adeno-associated virus (AAV). Replication of AAV in a host requires the assistance of host cells or other viruses, such as adenoviruses and herpesviruses, and thus are also referred to as adeno-associated viruses. AAV viral particles have a mean diameter of about 20 nm, an icosahedron, a molecular weight of about 5.5-6.2 x 106 daltons, and a density in CsCl gradients of 1.39-1.42 g/cm. Compared with other viruses, the adeno-associated virus is more stable and can bear the temperature of 60 ℃ and the action of an ion detergent. AAV genomes are linear single-stranded positive or negative strand DNA, approximately 4700 nucleotides in length. The genome of adeno-associated virus contains two open reading frames, rep and cap, with terminal repeats (ITRs) at each end.
The recombinant adeno-associated virus (rAAV) vector has the advantages of high expression efficiency in vitro and in vivo, long expression period, low immunity, no pathogenicity and tumorigenicity, low genetic toxicity, large-scale production and the like, so that the recombinant adeno-associated virus (rAAV) vector becomes a very promising biological vector in the current gene therapy field, and is widely applied to clinical experiments and achieves good curative effects.
It is well known that rAAV titer assays are critical to clinical trials. The field of gene therapy requires the establishment of standardized, sensitive and reliable titer assays to accurately assess clinical outcome and drug development using AAV. The current method for determining rAAV titer mainly detects the following aspects: viral particle titers (v.p.), genome titers (v.g.), infectious titers (Infectious Particles, i.p.), transduction titers (Transduction Units, t.u.), or enhanced transduction titers (Enhanced Transduction Units, e.t.u.). [ Clark, K.R., voulgaropoulou, F., fraley, D.M., and Johnson, P.R., cell lines for the production of recombinant adeno-associated viruses Hum.Gene Ther.1995,6, 1329-1341; clark K.R., liu, X., mcGrath, J.P., and Johnson, P.R., highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses.hum.Gene Ther.1999,10, 1031-1039 ]. qPCR has become the most widely used and accepted method for quantification of AAV vectors because it is simple and stable under ideal conditions, and it is rapid, sensitive and widely used. However, qPCR has its limitations, DNA amplification efficiency can be significantly compromised by various factors, including poor primer design, the presence of inhibitors or secondary structures in the template, and the requirement for an effective DNA standard curve, which can be incorrectly calibrated. Furthermore, for validation of the method, only two well-characterized reference standards are currently available for AAV serotypes 2 and 8. Because of the exponential amplification nature of the PCR reaction itself, and the potential for non-specific binding of primers, there is also a lack of stability and consistency in quantification.
The CRISPR/Cas system (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) is an acquired immune defense mechanism in prokaryotes against the invasion of foreign genes. Are evolving from bacteria and archaea in the process of protecting against the invasion of foreign viruses and phages. The system is capable of integrating a DNA fragment of a foreign invaded host into a CRISPR site and then guiding Cas endonuclease cleavage of the foreign DNA sequence by corresponding CRISPR RNAs (crRNAs) to resist virus or phage invasion.
With the deep mining of CRISPR systems, the trans-cleaving activity of Cas protein was found, cas12a protein also known as Cpf1 was found to have Ruv C endonuclease domain resembling Cas9, but no HNH domain, and the N-terminus of Cas12a did not have an α -helical recognition leaf, requiring no tracrRNA. When the system is in operation, the mature crRNA and Cas12a protein are combined to specifically identify the PAM sequence rich in T, and the PAM sequence is repaired after being cut to form a sticky end [ Yang Fancheng, wang William, zhang Jiaqiang, CRISPR-Cas12 and Cas13: the lesser-knownsiblings of CRISPR-Cas9[ J ]. Cell Biology and Toxicology, 2019, 35 (6): 489-492;Yan Winston X, hunnewell Pratyusha, alfonse Lauren E, et al Functionally diverse type V CRISPR-Cassystems [ J ]. Science, 2018, 363 (6422): 88-91]. Studies show that the Cas12a protein is smaller than the Cas9 protein, the crRNA is close to half of the total sg RNA of the Cas9, the crRNA enters cells more easily, and the experimental effect is better. The Cas12a protein has trans-cleavage activity, the Cas12a protein is specifically combined with targeted double-stranded DNA (dsDNA) under the guidance of crRNA and activates the trans-cleavage activity, indiscriminately cleaves nonspecific single-stranded DNA (ssDNA), fluorescent signals are emitted after ssDNA probe cleavage by modifying fluorescent groups and quenching groups at two ends of the ssDNA, and thus the accumulation of the fluorescent signals and the synchronization of target detection are realized, so that the reaction is monitored in real time.
With the analysis of the principle of CRISPR endonuclease such as Cas12a 'trans-cleavage' activity (trans-cleavage, or collateral effect), a new field of CRISPR detection with great innovation is developed. CN112980924 has disclosed a method for amplification-free measurement of DNA single molecules using Cas12a "trans-cleavage" activity, however, the method involves dd The PCR determination has higher requirements on detection instruments, and many research and development personnel cannot realize the detection in view of the lack of conditions in actual operation, so how to design a simpler, more convenient and easy-to-realize amplification-free titer determination method is still a problem to be solved in the field.
The contents of all articles and references cited herein are incorporated by reference in their entirety.
Disclosure of Invention
The application utilizes the characteristic that the target sequence of Cas12a is specifically combined to generate the bypass activity, and the application provides a novel method for directly testing the AAV genome titer without carrying out PCR exponential amplification, thereby avoiding systematic deviation and instability caused by exponential amplification and primer non-specific combination and rapidly, accurately and stably measuring the rAAV titer.
In one aspect, the application provides a method for measuring the titer of an amplification-free adeno-associated virus genome, comprising the steps of:
s1, preparing standard substances with different molar concentrations;
plasmid samples are used as quantitative standard substances, and the standard substances are packaged in batches in a specific storage solution at a lower concentration (10 times the uppermost concentration of standard curve quantification) for quantitative batch-to-batch stability, and are stored at-80 ℃ to prevent sample degradation. In some embodiments, the gradient of the molar concentration of the standard may be set to 3, 5,6, 10 or more depending on the case, and preferably, the gradient of the molar concentration of the standard may be set to 7.
S2, forming different Cas12a reaction systems by the standard substances with different molar concentrations, crRNA, cas12a protein and fluorescent DNA probes;
the Cas12a protein is Cas12a or a Cas protein having a bypass single-strand DNA cleavage activity similar to Cas12a, in particular, may be one or more of FnCas12a, asCas12a, enacas 12a, lbCas12a, lb5Cas12a, hkCas12a, osCas12a, tsCas12a, bbCas12a, boCas12a and Lb4Cas12 a; crrnas, i.e., guide RNAs, can guide Cas12a protein to specifically bind to a target nucleic acid molecule; the DNA probe, also referred to as a reporter, contains a detectable label, specifically, a fluorescent chromophore and a fluorescence quenching group can be labeled at both ends of the probe, respectively, and when the DNA probe is bypass-cleaved by Cas12a protein, a detectable signal is generated after the end with the fluorescent chromophore is separated from the fluorescence quenching group.
In some embodiments, the detectable signal of the DNA probe is a fluorescent signal.
In some embodiments, the DNA probe is non-specific.
In some embodiments, the DNA probe is a fluorescent reporter.
In some embodiments, the DNA probe is a single-stranded DNA labeled at both ends with a fluorescent chromophore and a fluorescence quenching group, respectively.
In some embodiments, the bases of the DNA probes are modified with modification sites at the 5 'end, 3' end, and/or middle of the probes.
In some embodiments, the base of the DNA probe is modified to tag a fluorescent chromophore HEX, JOE, TET, ROX, TAMRA, FAM at the 5' end.
In some embodiments, the base modification of the DNA probe is to label the fluorescence quenching groups BHQ1, BHQ2, MGB at the 3' end.
In some embodiments, the base modification of the DNA probe carries a labeled fluorescent chromophore FAM on one end and a fluorescence quenching group BHQ1 on the other end.
In some embodiments, the base of the DNA probe is modified to tag a fluorescent chromophore FAM at the 5 'end and a fluorescent quenching group BHQ1 at the 3' end.
In some embodiments, the DNA probe is a ssDNA-reporter, and the fluorescent chromophore HEX, JOE, TET, ROX, TAMRA, FAM is labeled at the 5' end of the ssDNA-reporter; and/or marking fluorescence quenching groups BHQ1, BHQ2 and MGB at the 3' end. Specifically, the modification of the base of the ssDNA-reporter is that a fluorescent luminous group FAM is marked at the 5 'end, a fluorescent quenching group BHQ1 is marked at the 3' end, and the base length of the ssDNA-reporter is 5 bases.
In some embodiments, the ssDNA-reporter has the structure 5'FAM-Nm-3' BHQ1, wherein N represents any base selected from A, T, C, G, 5'FAM represents FAM located at the 5' end, and 3'BHQ1 represents BHQ1 located at the 3' end; and m is a positive integer from 5 to 30, in particular m may be 5,6,7,8,9, 10,1,5, 20, 21, 22, 25, 30.
In some embodiments, the ssDNA-reporter has the structure 5'FAM-N21-3' BHQ1, wherein N represents any base selected from A, T, C, G, 5'FAM represents FAM located at the 5' end, and 3'BHQ1 represents BHQ1 located at the 3' end.
In some embodiments, the concentration of ssDNA-reporters is 0.5-10. Mu.M, preferably 1-5. Mu.M, more preferably 1-2. Mu.M, and most preferably 2. Mu.M.
In some embodiments, the standard needs to be pretreated prior to addition to the Cas12a reaction system; and (3) quantitatively digesting the purified rAAV by using nuclease, inactivating the rAAV, then carrying out proteinase K digestion, and inactivating the proteinase K to obtain a treated virus sample. Specifically, the nuclease is at least one of DNase I, DNase II and Benzonase nuclease, preferably the nuclease is DNase I and/or Benzonase nuclease, and more preferably the nuclease is DNase I nuclease. Specifically, the final concentration of proteinase K is 0.2 to 5 mg/ml, preferably 0.4 to 4 mg/ml, and more preferably 1 to 3 mg/ml; the treatment time of proteinase K is 0.15 to 4.4 h, preferably 0.25 to 3. 3 h, more preferably 0.5 to 2.2 h; the treatment temperature of proteinase K is 20 to 56 ℃, preferably 37 to 56 ℃, and more preferably 50 to 56 ℃.
In another embodiment, the final concentration of nuclease is 100U/ml; the nuclease treatment time was 0.5. 0.5 h; the nuclease treatment temperature was 37 ℃.
In another embodiment, the final concentration of proteinase K is 2 mg/ml; the treatment time of proteinase K was 0.5. 0.5 h; the treatment temperature of proteinase K was 55 ℃.
In another embodiment, the inactivation of nuclease and proteinase K includes heat inactivation, and other inactivation methods commonly used in the art may be employed.
S3, measuring fluorescence signals of different Cas12a reaction systems to prepare different fluorescence accumulation curves;
in the CRISPR detection stage, cas12a protein is combined with crRNA at 37 ℃ to form a Cas12a/crRNA complex, the Cas12a is specifically combined with a targeting sequence in a target fragment under the guidance of crRNA, the trans-cleavage activity of the Cas12a protein is activated, a non-specific ssDNA probe is indiscriminately cleaved, the fluorescent group and the quenching group are separated after the ssDNA probe modified with the fluorescent group and the quenching group is cleaved, the quenching group releases the blocking effect on the fluorescent group, and the change of the fluorescence intensity can be detected by means of a fluorescence signal detection device under the irradiation condition of light with a specific wavelength.
S4, obtaining different slopes according to the different fluorescence accumulation curves;
when the amount of Cas12a enzyme is much greater than the amount of target DNA in the standard (activator of Cas12a bypass activity) and the amount of probe (enzyme substrate) is much greater than Cas12a activated bypass activity, a certain amount of target DNA will activate a certain amount of bypass activity of Cas12a, the cleavage probe releases a fluorescent signal, the measured fluorescent signal will change linearly with time and the slope will remain unchanged over a certain range. Since the molar concentration of the amount of target DNA in the standards is different, the slope of the fluorescence accumulation curve varies for each group of standards under the same environment, i.e., with the total amount of enzyme, the crRNA, and the type and amount of probe remaining the same.
In order to makeThe slope is more accurate and significant, the application obtains the slope, selects the corresponding scattered points in the range of 5-30 min in the fluorescence accumulation curve, automatically generates a slope curve in the graph by Excel, and displays the equation and the R value. In some embodiments, calculating the slope within 5-30 min in the fluorescence accumulation curve corresponding to each standard, and selecting R 2 The slope with a value greater than 0.99 was further calculated.
S5, calculating a linear relation between the molar concentration and the slope of the standard substance to obtain a linear equation:
the standard substances with different molar concentrations can lead the reaction system to generate straight lines with different slopes in a fluorescent (y-axis) -reaction time (x-axis) coordinate system, and the molar concentrations of the standard substances and the slopes corresponding to the standard substances can form a linear relation, and a linear equation is obtained through calculation.
S6, forming a Cas12a reaction system by the sample to be detected, crRNA, cas12a protein and a fluorescent DNA probe, detecting a fluorescent signal of the reaction system, preparing a fluorescent accumulation curve and obtaining the slope of the sample to be detected;
s7, bringing the slope of the sample to be detected into the linear equation to obtain the molar concentration of the sample to be detected.
And (3) putting the unknown sample into the same environment as the standard sample, detecting a fluorescence signal of the unknown sample to obtain a fluorescence accumulation curve, and calculating the molar quantity of the fluorescence accumulation curve from a slope-molar quantity linear equation of the standard sample to obtain the titer of the recombinant adeno-associated virus.
The application discovers that the molar concentration of the adeno-associated virus in the reaction system and the slope of a fluorescence accumulation curve thereof can be linearly related by utilizing the Cas12a to bind with the adeno-associated virus by-pass fluorescence DNA probe, and further provides a titer detection method of the adeno-associated virus genome. The detection method does not need PCR amplification, is simple, convenient and easy to realize.
Further, the Cas12a Reaction system further includes a Buffer, and specifically, the Buffer may be a aurey 10×reaction Buffer.
Further, the crRNA is a crRNA targeting the ITR region of adeno-associated virus. The adeno-associated virus of the application can be natural or recombinant, and has ITR region. The AAV genome used in the examples is a linear single-stranded DNA of about 4.7. 4.7 kb comprising two open reading frames upstream and downstream, rep and Cap, and 2 Inverted Terminal Repeats (ITRs) consisting of 145 nucleotides, and specifically the crRNA sequence is as shown in SEQ ID NO. 3. The crRNA provided by the application has a larger dynamic range in a Cas12a reaction system, and is favorable for good differentiation of standard substances or samples to be detected with different concentrations.
Further, the sequence of the single-stranded DNA probe is shown as SEQ ID NO. 4. The DNA probe provided by the application has obvious differentiation between the fluorescence value measured in the Cas12a reaction system and the background fluorescence value, and is favorable for distinguishing the standard substance or the sample to be detected from the background signal under different concentrations.
Further, the molar ratio between the standard and Cas12a protein and single-stranded DNA probe is in the range of 10 -5 ~10 -2 :0.5 to 1.5: 2-5, wherein 10 -5 10 -2 Are all in magnitude units, in particular, 10 -5 Represents x 10 -5 X represents any number between 0 and 10,10 -2 Represents x 10 -2 X represents any number between 0 and 10.
In some embodiments, the molar ratio between the standard and Cas12a protein and single-stranded DNA probe ranges from 10 -4 ~10 -2 :0.5 to 1.5: 2-5. Specifically, 10 -5 Represents x 10 -4 X represents any number between 0 and 10,10 -2 Represents x 10 -2 X represents any number between 0 and 10.
In some embodiments, preferably, the molar ratio between the standard and Cas12a protein and single-stranded DNA probe ranges from 3 x 10 -4 ~3*10 -2 ：1：4。
In some embodiments, preferably, the molar ratio between the standard and Cas12a protein and single-stranded DNA probe ranges from 3.57×10 -4 ~2.29*10 -2 ：1：4。
The application provides a detection system which selects theThe molar ratio of the standard substance of the crRNA and the single-stranded DNA probe provided by the application to the Cas12a protein and the single-stranded DNA probe is in the range of 10 -5 ~10 -2 :1: and when the gradient is within 4, the gradient of the standard substance is stable in growth and easy to read, the gradients of the standard substances with different molar concentrations are easy to distinguish, and the linear equation obtained through the gradients can accurately and stably measure the titer.
In some embodiments, in the case where the concentration of single-stranded DNA probe is much greater than Cas12a protein, the molar ratio of standard to single-stranded DNA probe should be maintained within a certain range to ensure that the fluorescence accumulation curve remains slope unchanged within a certain range and is easy to read and distinguish. Specifically, the molar ratio of standard to single-stranded DNA probe was 3.57×10 -4 ~2.29*10 -2 ：4。
In another aspect, the present application provides a crRNA, specifically, a sequence of crRNA as shown in SEQ ID No.3, which can generate a higher fluorescence value and a lower background fluorescence value, i.e., have a larger dynamic range, in the Cas12a reaction system.
On the other hand, the application provides a single-stranded DNA probe, and specifically, the sequence of the single-stranded DNA probe is shown as SEQ ID NO.4, the difference between the fluorescence value and the background fluorescence value in a Cas12a reaction system is remarkable, and the single-stranded DNA probe is favorable for distinguishing a standard substance or a sample to be detected from a background signal under different concentrations.
In some embodiments, the application provides a kit for adeno-associated virus genome comprising crRNA having a sequence as shown in SEQ ID NO.3 and a single-stranded DNA probe having a sequence as shown in SEQ ID NO. 4.
In some embodiments, the sequence shown in SEQ ID No.3 is used as crRNA, which has a large dynamic range in Cas12a reaction system, thus making the fluorescence accumulation curves of standards of different molar concentrations have a large difference.
In some embodiments, the sequence shown in SEQ ID No.4 is used as a fluorescent single-stranded DNA probe, and the fluorescence value and the background fluorescence value in the Cas12a reaction system are remarkably different, so that the standard substance or the sample to be detected can be well distinguished from the background signal under different concentrations.
In some embodiments, the sequence shown in SEQ ID NO.3 is used as crRNA, the sequence shown in SEQ ID NO.4 is used as fluorescent single-stranded DNA probe, and the molar ratio between the standard and the Cas12a protein and single-stranded DNA probe is in the range of 10 -5 ~10 -2 :1:4, the application provides crRNA with larger dynamic range, so that fluorescence accumulation curves between standards with different molar concentrations have larger difference; the single-stranded DNA fluorescent probe with obvious difference between the fluorescent signal and the background signal is provided, so that a standard substance or a sample to be detected can be well distinguished from the background signal under different concentrations; according to the application, the fluorescence accumulation curve of the standard substance is kept unchanged in a certain range by providing the range of the molar ratio between the standard substance and the Cas12a protein as well as the single-stranded DNA probe, and under the conditions of the crRNA and the fluorescence single-stranded DNA probe provided by the application, the slopes of the fluorescence accumulation curves displayed by the standard substances with different molar concentrations in a larger concentration range are easy to read and distinguish, so that a more accurate linear relation is obtained.
The titer measurement method of the adeno-associated virus genome provided by the application comprises the steps of preparing standard substances with different molar concentrations; the standard substances with different molar concentrations, crRNA, cas12a protein and DNA probes form different Cas12a reaction systems; measuring fluorescence signals of different Cas12a reaction systems to prepare different fluorescence accumulation curves; obtaining different slopes according to the different fluorescence accumulation curves; calculating the linear relation between the molar concentration and the slope of the standard substance to obtain a linear equation: forming a Cas12a reaction system by a sample to be detected, crRNA, a Cas12a protein, a fluorescent luminescent group and a fluorescent quenching group double-labeled single-stranded DNA probe, detecting a fluorescent signal of the reaction system, preparing a fluorescent accumulation curve and obtaining a slope of the sample to be detected; and carrying the slope of the sample to be detected into the linear equation to obtain the molar concentration of the sample to be detected, thereby obtaining the titer. The test method not only does not need PCR amplification, but also has low requirements on test instruments, and the titer of the adeno-associated virus genome can be simply, rapidly, accurately and stably measured by using a common qPCR instrument.
Drawings
For a better understanding of the application and to show more clearly how it may be carried into effect, reference will now be made, by way of example, to the accompanying drawings and illustrate features according to embodiments of the application, in which:
fig. 1: schematic diagram of recombinant AAV structure.
Fig. 2: the method for determining recombinant adenovirus titer by qPCR comprises the steps of (A) amplifying a curve and (B) melting the curve by qPCR from standard plasmid with known serial dilution concentration.
Fig. 3: crRNA screening results. Four sets of Cas12a reaction systems were set up, respectively, and fluorescence accumulation curves measured for crRNA1, crRNA2, crRNA3, and blank control under other conditions of reaction were consistent.
Fig. 4: three groups of Cas12a reaction systems are respectively arranged, and fluorescent accumulation curve results are measured on 5nt, 10nt and 21nt fluorescent DNA probes under the condition that other reaction conditions are consistent.
Fig. 5: and 7 groups of Cas12a reaction systems are respectively arranged, and fluorescent accumulation curve results measured on AAV7 and AAV8 of standard products and to-be-measured products with different molar concentrations under the condition that other reaction conditions are consistent.
FIG. 6; in the Cas12a reaction system, the application range of the standard substance, namely the molar ratio between the standard substance and Cas12a protein and single-stranded DNA probe is 2.86 x 10 -2 :1:4 (n=2); the molar ratio between the standard and Cas12a protein and single-stranded DNA probe was 2.86 x 10 -4 :1:4 (n=2), the fluorescence accumulation curve of the Cas12a reaction system; n=number of repetitions per group.
Fig. 7: standard curves drawn at a single time point (5 min) were drawn through Cas12a reaction system fluorescence accumulation curves of different molar concentration standards.
Fig. 8: standard curves drawn by slopes of fluorescence accumulation curves of Cas12a reaction systems of different molar concentration standards. The horizontal axis is the calculated slope, the vertical axis is the molar quantity of the standard substance, the standard curves of the two are calculated according to the slope and the standard substance, and the molar quantity of the template DNA in the unknown sample can be calculated from the slope-molar quantity standard curve of the standard substance, so that the titer of the recombinant adeno-associated virus is obtained.
Detailed Description
Definition: in order to provide a clear and consistent understanding of the terms used in the description of the present application, some definitions are provided below. Furthermore, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
The word "comprising" (and any form of comprising, such as "comprising" and "comprises"), "having" (and any form of having, "having", "including" and "containing") as used in this specification and claims is inclusive and open-ended and does not exclude additional unrecited elements or process steps.
The terms "about" or "approximately" are used to indicate that the value includes errors in the instruments and methods used in determining the value. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
The present specification relates to a number of terms and abbreviations used by those skilled in the art. However, for the sake of clarity and consistency, definitions of selected terms are provided.
The terms "Cas12a", "Cas12a protein" or "Cas12a enzyme" are used interchangeably to refer to a class of endonucleases in a CRISPR system, such as enacas 12a (a variant of Cas12 a), cas12a being characterized by both cis and trans single stranded DNA cleavage activity.
The term "crRNA" is used to refer to a small guide molecule that is capable of directing targeting a specific nucleic acid molecule and activating Cas12a cleavage activity.
The term "target fragment" refers to a region of a DNA molecule that is specifically recognized and bound by a guide RNA (gRNA) in crRNA. The guide sequence is typically about 17-24 bases, typically 20 bases, at the 5' end of the crRNA. In addition, the target fragment should also include PAM sequences recognized by Cas12a, such as TTTV (V represents adenine, guanine or thymine).
"DNA standard" is used herein to mean a DNA molecule, such as a recombinant DNA plasmid, comprising the above-described target fragment at a concentration. To obtain a linear relationship between the molar concentration of the DNA standard and the slope of the fluorescence accumulation curve, the DNA standard is typically formulated (or diluted) to a plurality of different concentrations.
The terms "fluorescent DNA probe", "reporter" or "ssDNA-reporter" are used interchangeably to refer to a length of single stranded DNA (ssDNA) to which a fluorescent group and a quenching group are added to the bases at the 5 'and 3' ends. The function of the fluorescent dye in the CRISPR detection system is that after the ssDNA is nonspecifically cut by the Cas12a, the fluorescent group is separated from the quenching group, the quenching group releases the blocking effect on the fluorescent group, and the change of the fluorescence intensity can be detected by means of a fluorescence signal detection device under the irradiation condition of light with a specific wavelength. In some embodiments, the length of the fluorescent DNA probe is 5-21nt, specifically 2, 10, 21, preferably the length of the fluorescent DNA probe provided by the present application is 5nt.
The term "slope" is used to denote the amount by which a straight line (or tangent to a curve) is inclined with respect to the (transverse) coordinate axis. It is usually expressed by the tangent of the angle of a straight line (or tangent of a curve) to the (abscissa) axis, or the ratio of the difference between the ordinate of the two points to the difference between the abscissa. Because the standard substance provided by the application is generally a curve of a fluorescence accumulation line measured by reaction in a qPCR instrument, and the slope can be kept unchanged for a certain time under certain environments or conditions, the slope is the slope displayed by the standard substance in a time period when the slope is kept unchanged. The term "slope remains unchanged" shall mean that the slope remains unchanged as a whole, or that the slope changes within a small range of magnitudes, not to mean that the slope is completely unchanged.
Examples
The application will be more readily understood by reference to the following examples, which are provided to illustrate the application and should not be construed to limit the scope of the application in any way.
Unless defined otherwise or the context clearly indicates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application.
All materials or instruments used herein are commercially available unless otherwise defined or the context clearly dictates otherwise.
Although the present application has been described in detail with reference to the embodiments thereof, these embodiments are provided for the purpose of illustration and not limitation of the application. Other embodiments that can be obtained according to the principles of the present application fall within the scope of the application as defined in the claims.
Example 1: design of crRNA
Three crrnas were designed based on the ITR region target sequence in AAV, crRNA1, crRNA2 and crRNA3, respectively, with the crRNA sequences shown in table 1. The components of the detection system are shown in Table 2 below. After the system preparation is completed, the eight-connecting tube is placed in a fluorescent quantitative PCR instrument to react for 60 min at 37 ℃, fluorescence value data are read, after the reaction is completed, the eight-connecting tube is used for agarose gel electrophoresis, and the cut strips are observed under a gel imaging system.
The crRNA design results are shown in fig. 3, and fig. 3 is a fluorescence curve measured by a qPCR instrument, and it can be seen from the graph that crRNA3 can generate a higher fluorescence value and the set of background fluorescence values is relatively lower, so crRNA3 is selected as crRNA in the Cas12a detection system. The fluorescence value change range of the crRNA3 in the Cas12a detection system is obviously better than that of the crRNA1 and the crRNA2, so that the condition that the noise is too large to cover the real level of the Cas12a system is avoided, the standard product can show larger difference under different concentrations, and further, a more accurate linear relation is obtained.
Example 2: design of ssDNA probes
To achieve the best analytical performance of the detection system, three different ssDNA probes, namely 5nt, 10nt and 21nt, were designed in the CRISPR detection system, the sequences of which are shown in table 3. Under the condition that the final concentration of ssDNA probes in a system is kept the same, after detection preparation of plasmids containing Cas12a protein, crRNA3, target sequences, buffer solution and different ssDNA probes is completed, the plasmids are fully mixed, then eight connecting tubes are placed in a fluorescent quantitative PCR instrument for reaction at 37 ℃ for 60 min, fluorescence value data are read, and after the reaction is completed, the obtained products are used for agarose gel electrophoresis and bands after cutting are observed under a gel imaging system. Finally, the ssDNA probe with better effect is designed.
As shown in FIG. 4, it can be seen from the graph that although the relative fluorescence values measured by the ssDNA probes of ssDNA-reporter-10nt and ssDNA-reporter-21nt are higher, the background signals are also relatively higher, which is unfavorable for distinguishing samples at different molar concentrations, while the ssDNA probes of ssDNA-reporter-5nt have higher relative fluorescence values and lower backgrounds than other ssDNA probes, so that the standard or the sample to be measured can be well distinguished from the background signals at different concentrations, and further a more accurate linear relationship is obtained.
Example 3: construction of Cas12a fluorescence detection platform
The brief procedure is as follows:
1) DNase I treatment: purified rAAV samples were treated with DNaseI enzyme (1U/. Mu.1), digested at 37℃for 30 minutes, reacted at 95℃for 10 minutes to inactivate the digested surrounding DNA, and AAV samples provided herein were AAV7 and AAV8, respectively.
2) Proteinase K treatment: purified rAAV samples were treated with proteinase K (20 mg/mL), digested at 55deg.C for 30 min to remove viral coat, then reacted at 95deg.C for 10 min to inactivate, centrifuged at 12000 rpm for 2 min, and the supernatant was placed in a centrifuge tube at 1.5 mL.
3) Preparing a standard substance solution: the standard plasmid was diluted to 2.29×10 with sterile water -9 mol/L, and then sequentially diluting with sterile water to 2.29×10 -9 mol/L、1.14×10 -9 mol/L、5.71×10 -10 mol/L、2.86×10 -10 mol/L、1.43×10 -10 mol/L、7.14×10 -11 mol/L、3.57×10 -11 mol/L, 7 gradients total;
4) Cas12a cleavage reaction: in a 20 μl reaction system, cas12a detection system was formulated with the following components: the quantitative PCR Reaction system was added with standard (10. Mu.1) and rAAV (10. Mu.1) at each concentration treated in step 3, synthesized crRNA3 (0.1. Mu.M), cas12a (0.5. Mu.M), ssDNA-reporter-5nt (FAM-N5-BHQ 1, 2. Mu.M), and Buffer solution of gold 10X Reaction Buffer, as specified in Table 4 below. The reaction was terminated by reacting at 37℃for 30-60 min and then at 98℃for 2 min.
3 repeated holes are respectively arranged on the series concentration standard substance and the recombinant adeno-associated virus sample to be detected; adding the prepared system (without template) into a quantitative PCR plate, and adding templates in sequence; the edges of each hole are sealed by tightly adhering to the sealing plate film so as to prevent the PCR reaction liquid from being volatilized by heating.
The fluorescent quantitative PCR instrument was run with the parameters set forth in Table 5 below, with a reaction time of 30-60 min, a cycle of one cycle per minute was set, and a fluorescent value was recorded:
。
the results are shown in FIG. 5, which shows the fluorescence accumulation curves obtained in the qPCR instrument for the standard plasmid and the sample to be tested.
According to the titer detection method of the adeno-associated virus, provided by the application, the slope of the fluorescence accumulation growth curve of the standard substance with different molar concentrations in the Cas12a system is required to be measured, and then the linear relation is obtained through different slopes corresponding to different molar concentrations. Therefore, the detection results of the standard substances with different molar concentrations are obviously distinguished, and the slope of the fluorescence accumulation growth curve of the standard substance or the sample to be detected is basically kept unchanged within the time range of 5-30 min, so that a more accurate slope is obtained.
The application discovers that when the enzyme quantity of the Cas12a is far greater than that of the DNA to be detected (an activator of the bypass activity of the Cas12 a) and the probe quantity (enzyme substrate) is far greater than that of the Cas12a activated by the bypass activity, a certain quantity of the DNA to be detected activates a certain quantity of bypass activity of the Cas12a, the fluorescent signal released by the probe is cut, the measured fluorescent signal is linearly changed along with time within a certain time range, and the slope is kept unchanged.
The application also finds that the molar ratio between the DNA to be tested and Cas12a enzyme and probe must be maintained within a certain range (ensuring that the amount of enzyme and probe is much greater than that of the target DNA, but also that enough enzyme needs to be activated to generate a fluorescent signal sufficient for detection) to ensure the stability and variability of the slope. As can be seen from FIG. 6, when the ratio between the standard and the Cas12a enzyme and single-stranded DNA probe is too low, i.e., the molar concentration of the standard is 2.86×10 -11 At mol/L, the fluorescence signal is too small, almost as same as background (control without standard substance) does not increase cumulatively with time (the slope is approximately equal to zero), and the fluorescence signal is greatly influenced by noise, so that the correct slope can not be obtained; when the ratio between the standard and Cas12a enzyme and single-stranded DNA probe is high, i.e. the molar concentration of the standard is 2.86 x 10 -9 When the molecular weight is mol/L, fluorescence of the fluorescent material increases rapidly, the fluorescent material reaches a plateau stage rapidly, scattered points with stable trend cannot be obtained in a time range of 5-30 min, and then an effective slope cannot be obtained. Thus the concentration of the standard should be within a certain range.
Different molar amounts of standard will cause the reaction system to produce lines of different slopes in the fluorescent (y-axis) -reaction time (x-axis) coordinate system. The fluorescence accumulation diagrams corresponding to the standard substances and the to-be-detected substances with different molar concentrations are shown in fig. 5, the slopes are shown in tables 6 and 7, and the slope-molar quantity standard curve of the standard substance is shown in fig. 8. The molar quantity of the template DNA in the unknown sample can be calculated from the slope-molar quantity standard curve of the standard substance, so as to obtain the titer of the recombinant adeno-associated virus.
The calculation formula is as follows:。
n: molar quantity of recombinant adeno-associated virus sample to be tested
NA: avogalileo constant
V: volume of recombinant adeno-associated virus sample to be tested
。
。
The viral titers of AAV7 and AAV8 were calculated as 2.15X10, respectively, by the straight line obtained by slope regression 12 （vg/mL）、1.41×10 13 （vg/mL）。
In addition, the application also provides another method for measuring titer, namely a point measurement method, namely, the fluorescent values of the standard substances with different concentrations at a certain time point shown in the figure 5 are read, then the corresponding linear relation is calculated according to the molar concentration and the corresponding fluorescent values, the corresponding fluorescent values when the system is adopted to react for five minutes are shown in the figure 7, the regression obtained linearity of the method is not as good as that obtained by the slope method (R 2 The value is less than 0.99), and the error is larger. The titer of AAV7 of the sample to be tested was calculated to be 7.34×10 using these regression equations 11 (vg/mL) the titer of AAV8 in the sample to be tested was 2.49X10 12 (vg/mL). These titers obtained using fixed point regression are also significantly lower than those calculated using slope regression.
Comparative example: qPCR determination of titers of recombinant adeno-associated viruses
In order to evaluate the accuracy of the AAV virus titer detection method provided by the application, the application provides a conventional qPCR method for detecting the titer of a sample to be detected as a positive control, and the specific steps are as follows:
1. sample dilution: the 5 mu 1 virus stock was added to 45 mu 1 PBS+PF68 (0.1M NaCl) and mixed by pipetting.
2. DNase I treatment: purified rAAV samples were treated with DNaseI enzyme (1U/. Mu.1), digested at 37℃for 30 minutes and then reacted at 95℃for 10 minutes to inactivate the digested surrounding DNA, and AAV samples provided herein were AAV7 and AAV8, respectively.
3. Proteinase K treatment: purified rAAV samples were treated with proteinase K (20 mg/mL), digested at 37℃for 30 min, inactivated at 95℃for 10 min to remove viral capsids, centrifuged at 12000 rpm for 2 min, and the supernatants were placed in centrifuge tubes at 1.5: 1.5 mL.
4. quantitative qPCR calculation: preparing a standard substance solution: the standard plasmid (containing the recombinant adenovirus specific primer amplified sequences) was diluted to 5X 10 per microliter with sterile water 9 Copy number (10-fold gradient dilution with sterile water, followed by 5X 10 dilution in sequence 8 、5×10 7 、5×10 6 、5×10 5 、5×10 4 、5×10 3 、5×10 2 0 copies/. Mu.1 total of 8 gradients;
3 repeated holes are respectively arranged on the series concentration standard substance and the recombinant adeno-associated virus sample to be detected; adding the prepared system (without template) into a quantitative PCR plate, and adding templates in sequence; the sealing plate film is tightly adhered to ensure that the edge of each hole is sealed so as to prevent the PCR reaction liquid from being volatilized by heating; the quantitative PCR reaction system is shown in Table 8.
The fluorescent quantitative PCR instrument was run at the parameter settings shown in table 9 below:
。
the results are shown as a and B in fig. 2, showing amplification and melting curves obtained from qPCR of standard plasmids of known serial dilution concentration (copy number); copies of the template sample within a certain rangeThe logarithmic number (content or concentration) and Ct value measured by qPCR are linearly related. And calculating the logarithm of the copy number of the template according to a standard curve, so as to calculate the titer of the recombinant adeno-associated virus to be detected, and the number of virus particles (vg/ml) =relative value of standard substance multiplied by dilution. The virus titers of AAV7 and AAV8 were calculated to be 4.44X10, respectively 12 （vg/mL）、1.14×10 13 （vg/mL）。
The three methods for measuring the titer of AAV viral genomes adopted by the application have the measurement results shown in the following table 10:
as can be seen from the above, AAV titers measured by the dot extraction method are obviously different in magnitude orders from those measured by the conventional fluorescence quantitative PCR method, but the titers measured by the method provided by the application are almost indistinguishable from those measured by the conventional method, and the CV value is lower than that of the titers measured by the conventional method, so that the result is more stable. The method provided by the application does not need PCR amplification, has low requirements on a testing instrument, and can simply, rapidly, accurately and stably determine the titer of the adeno-associated virus genome by using a common qPCR instrument.
Although the present application has been described in detail with reference to the embodiments thereof, these embodiments are provided for the purpose of illustration and not limitation of the application. Other embodiments that can be obtained according to the principles of the present application fall within the scope of the application as defined in the claims.
Claims (3)
1. An amplification-free adeno-associated virus titer measurement method comprising the steps of:
s1, preparing DNA standard products with different molar concentrations;
s2, the DNA standard products with different molar concentrations respectively form different DNA standard product Cas12a reaction systems with crRNA, cas12a protein and DNA probes targeting target fragments in the DNA standard products;
s3, measuring fluorescence signals of the different Cas12a reaction systems along with time change to prepare different fluorescence accumulation curves;
s4, obtaining different slopes according to the different fluorescence accumulation curves;
s5, obtaining a linear equation representing the relation between the DNA standard substance molar concentration and the slope according to the DNA standard substance molar concentration and the slope:
s6, forming a reaction system of the sample to be detected Cas12a by the sample to be detected comprising the adeno-associated virus genome, the crRNA, the Cas12a protein and the DNA probe, detecting a fluorescent signal of the reaction system of the sample to be detected, preparing a fluorescent accumulation curve and obtaining a slope of the sample to be detected;
s7, substituting the slope of the sample to be detected into the linear equation to obtain the molar concentration of the adeno-associated virus genome in the sample to be detected, thereby obtaining the titer of adeno-associated virus in the sample to be detected,
wherein the target fragment is from the adeno-associated virus genome,
wherein the DNA probe is a single-stranded DNA probe comprising a fluorescent group and a fluorescence quenching group,
wherein the sequence of the crRNA is shown as SEQ ID NO.3,
wherein the sequence of the DNA probe is shown as SEQ ID NO.4,
wherein the molar ratio between the different molar concentrations of the standard and the Cas12a protein and the DNA probe is: 10 -5 ~10 -2 ：0.5~1.5：2~5。
2. The measurement method of claim 1, wherein the Cas12a protein is one or more of FnCas12a, asCas12a, enacas 12a, lbCas12a, lb5Cas12a, hkCas12a, osCas12a, tsCas12a, bbCas12a, boCas12a, and Lb4Cas12 a.
3. The method of measurement according to claim 1, wherein the target fragment is from the ITR region of the adeno-associated viral genome.
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