CRISPR-Cas12a-based chromosome aneuploid rapid detection method before embryo implantation

Abstract

The invention relates to the technical field of molecular biological detection, in particular to a method for rapidly detecting chromosome aneuploidy before embryo implantation based on a CRISPR-Cas12a (clustered regularly interspaced short palindromic repeats-associated 12a) system. The method comprises the following steps: acquiring a biopsy cell sample, processing the sample to release DNA, preparing a CRISPR-Cas12a detection reaction system, adding the sample DNA and carrying out a reaction, detecting fluorescence signal change, calculating a fluorescence signal ratio of a to-be-detected chromosome to an internal reference chromosome, finally judging a chromosome copy number state according to the signal ratio, and obtaining a result from the sample and outputting the result. The method only needs 2-4 hours, so that aneuploid screening can be completed before embryo transplantation when a fresh embryo transplantation strategy is adopted, the potential influence of embryo freeze thawing on quality is reduced, a common fluorescence detector instead of an expensive high-throughput sequencer or a microarray scanner is used, and the single sample detection cost is reduced by 50-70%.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology detection technology, and more specifically, to a method for rapid detection of preimplantation chromosomal aneuploidy based on a CRISPR-Cas12a system, which is particularly suitable for preimplantation genetic testing (PGT-A) in assisted reproductive technology, for screening abnormalities in embryo chromosome numbers and assisting in selecting embryos with normal chromosome composition for transplantation. Background Art

[0002] Chromosomal aneuploidy refers to the state in which the number of chromosomes in cells deviates from the normal diploid state, including chromosome deletion (monosomy) or extra chromosomes (trisomy), which is the main cause of early embryo arrest, miscarriage and congenital birth defects. The most common chromosomal aneuploidies include trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), trisomy 13 (Patau syndrome), etc. In order to reduce the risk of giving birth to an abnormal fetus, preimplantation genetic testing (PGT-A) technology is usually used in the process of assisted reproduction to perform chromosome screening on in vitro fertilized embryos to identify chromosomal aneuploid embryos.

[0003] Currently, the PGT-A technologies commonly used in clinical practice mainly include fluorescence in situ hybridization (FISH), chromosome microarray analysis (aCGH), real-time quantitative PCR (qPCR) and next-generation sequencing (NGS). Although these methods have been widely used in clinical practice, they still have the following significant shortcomings:

[0004] First, the traditional PGT-A technology has a long detection cycle, usually requiring 24-72 hours to complete the detection process. For example, the NGS method requires multiple steps such as library construction, sequencing, and data analysis, and the aCGH method requires time-consuming steps such as sample hybridization and washing. This long detection cycle significantly prolongs the in vitro culture time of embryos and increases the need for embryo freezing-thawing, which may have an adverse effect on embryo quality and implantation potential. Especially in the "fresh embryo transfer" clinical program, it is crucial to quickly obtain test results.

[0005] Secondly, existing technologies are expensive. Technologies such as NGS and aCGH require expensive high-throughput sequencers or microarray scanners and other professional equipment, as well as complex reagent systems. The cost of a single test is as high as 1,000-2,000 yuan, which limits their promotion and application in primary medical institutions and developing countries.

[0006] Third, the technical threshold is high. Existing technologies require complex sample processing and data analysis, and require high professional skills of operators. For example, NGS technology requires professional operations such as library construction, double-end sequencing, and bioinformatics analysis; aCGH technology requires complex steps such as labeling, hybridization, and image analysis. Most IVF laboratories lack such professionals and need to send samples to professional genetic laboratories, which increases the risk and time cost of sample transportation.

[0007] Fourth, the challenge of detection sensitivity. Embryo biopsy samples usually contain only 5-10 cells and very little DNA (about 30-60pg), which requires steps such as whole genome amplification (WGA), which may introduce amplification bias or errors and affect detection accuracy. In a laboratory environment with limited resources, how to ensure the detection sensitivity of trace samples is a key challenge.

[0008] In recent years, nucleic acid detection technology based on the CRISPR-Cas system has attracted widespread attention due to its high specificity, sensitivity and simplicity. The CRISPR-Cas12a (also known as Cpf1) system is an RNA-guided DNA endonuclease that is activated and acquires non-specific cleavage activity (collateral cleavage) on single-stranded DNA after recognizing and binding to target DNA. This feature provides a new technical platform for nucleic acid detection and has demonstrated application potential in the fields of infectious disease diagnosis and genetic disease screening.

[0009] In 2018, Chen et al. developed the DETECTR system (DNA Endonuclease Targeted CRISPRTrans Reporter), which used the side chain cleavage activity of Cas12a to detect specific nucleic acid sequences; in 2020, Doudna's team applied a similar principle to the rapid detection of the new coronavirus. However, the technical solution for applying the CRISPR-Cas12a system to the rapid detection of preimplantation chromosomal aneuploidy has not been reported. The development of a rapid, sensitive, simple and economical method for detecting preimplantation chromosomal aneuploidy based on the characteristics of the CRISPR-Cas12a system has important clinical needs and application value. Summary of the invention

[0010] The purpose of the present invention is to provide a method for rapid detection of pre-implantation chromosomal aneuploidy based on CRISPR-Cas12a, which can significantly shorten the detection time, reduce the technical threshold and detection cost, while maintaining high sensitivity and specificity, and is suitable for clinical IVF laboratory environments, providing a new technical solution for rapid screening of embryonic chromosomal aneuploidy.

[0011] To achieve the above object, the present invention provides the following technical solutions:

[0012] To achieve the above objectives, the present invention provides a method for rapid detection of preimplantation chromosomal aneuploidy based on CRISPR-Cas12a, the method comprising the following steps: a) obtaining a biopsy cell sample from an embryo; b) processing the biopsy cell sample to release genomic DNA; c) preparing a CRISPR-Cas12a detection reaction system, the reaction system comprising a Cas12a protein, a crRNA for a chromosome to be detected, a crRNA for an internal reference chromosome, and a fluorescent reporter substrate; d) adding the treated sample DNA to the CRISPR-Cas12a detection reaction system and reacting under appropriate conditions; e) detecting and recording changes in fluorescence signals; f) calculating the fluorescence signal ratio of the chromosome to be detected to the internal reference chromosome; and g) judging the copy number status of the chromosome to be detected according to the signal ratio.

[0013] The basic principle of the present invention is based on two key characteristics of the CRISPR-Cas12a system: 1) Sequence-specific recognition: Cas12a protein specifically recognizes and binds to the target DNA sequence under the guidance of crRNA; 2) Collateral cleavage activity: Cas12a is activated after binding to the target DNA and obtains the activity of cutting single-stranded DNA. Figure 2 , the CRISPR-Cas12a system works as follows: crRNA guides Cas12a protein to recognize and bind to a target DNA sequence with a specific PAM sequence (5'-TTTV-3', where V is A, C or G); after binding, Cas12a is activated and cuts the target DNA; at the same time, the activated Cas12a obtains non-specific cutting activity for single-stranded DNA, and the added single-stranded DNA reporter substrate with fluorophore and quencher is cut to produce a fluorescent signal. By designing crRNA for specific chromosome sequences, detection of specific chromosomes can be achieved. When the target chromosome copy number increases (such as trisomy), the number of activated Cas12a increases accordingly, producing a stronger fluorescent signal; conversely, when the target chromosome copy number decreases (such as monomer), the fluorescent signal weakens.

[0014] The technical solution of the present invention innovatively applies the CRISPR-Cas12a system to chromosome aneuploidy detection, which not only solves the core problems of long detection cycle, high cost, and complex operation, but also provides a sensitive and reliable detection platform with the following significant advantages:

[0015] 1. Significantly shortened detection cycle: From sample acquisition to result output, this method only takes 2-4 hours, which is about 90% more time efficient than the 24-72 hours of the traditional NGS / aCGH method. This enables aneuploidy screening to be completed before embryo transfer when a fresh embryo transfer strategy is adopted, reducing the potential impact of embryo freezing and thawing on quality.

[0016] 2. Significantly reduced costs: This method uses an ordinary fluorescence detector instead of an expensive high-throughput sequencer or microarray scanner, with low reagent costs. The cost of single sample testing is reduced by 50-70%, greatly improving the economic efficiency of clinical applications.

[0017] 3. Easy to operate: This method simplifies the sample processing steps, reduces complex experimental operations, and reduces the requirements for the professional skills of operators. The operation steps are reduced from 8-12 steps in the traditional method to 3-5 steps, so that routine IVF laboratory personnel can master it after simple training.

[0018] 4. High detection sensitivity: This method uses the side chain cleavage activity of Cas12a for signal amplification. A single activated Cas12a can cleave multiple substrate molecules, so that the detection limit reaches about 10pg DNA, which is suitable for trace sample detection.

[0019] 5. Good specificity: Through optimized crRNA design and multi-point targeting strategy, this method achieves a specificity of ≥98% and can accurately distinguish different chromosome copy number states.

[0020] 6. Strong compatibility: This method can be seamlessly integrated with the existing IVF laboratory workflow, eliminating the need to send samples externally, reducing sample transportation risks and time costs.

[0021] 7. Wide application scenarios: This method is not only suitable for conventional PGT-A testing, but can also be extended to multiple fields such as non-invasive embryo testing, subchromosomal variation detection, and chromosome structural abnormality detection.

[0022] The above advantages make the present invention a significant technological advancement in clinical applications and provide new technical support for improving the success rate of assisted reproductive technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure is a schematic diagram of the overall process of the method for rapid detection of pre-implantation chromosome aneuploidy based on CRISPR-Cas12a of the present invention.

[0024] Figure 2 Schematic diagram of the working principle of the CRISPR-Cas12a system in recognizing the target sequence and producing side chain cleavage activity after being activated.

[0025] Figure 3This is a schematic diagram of the principle of the multi-color multiplex detection technology in the present invention, showing how to simultaneously detect multiple chromosomes in the same reaction system.

[0026] Figure 4 This is a fluorescence signal curve diagram of detecting chromosome 21 trisomy using the basic single-tube method in Example 1, wherein A is a normal diploid sample, B is a trisomy 21 sample, and C is a monosomy 21 sample.

[0027] Figure 5 This is a multi-channel fluorescence signal diagram of simultaneously detecting chromosomes 21, 18, 13, X, and Y using a multi-color multiplex detection method in Example 2.

[0028] Figure 6 This is a result analysis diagram of detecting subchromosome level variation using droplet digital CRISPR-Cas12a technology in Example 3.

[0029] Figure 7 This is a comparison chart of the detection results of the method of the present invention and the traditional NGS method, showing the consistency of the two methods.

[0030] Figure 8 This is a comparison chart of the detection time, cost and accuracy of the method of the present invention with those of the prior art. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is described in detail below through specific embodiments.

[0032] Example 1: Basic single-tube CRISPR-Cas12a aneuploidy detection

[0033] This embodiment provides a method for detecting trisomy 21 (Down syndrome) by a single-tube method based on the CRISPR-Cas12a system, which uses LbCas12a protein and its corresponding crRNA to determine the chromosome copy number status by changes in fluorescence signals.

[0034] In this embodiment, first, a trophectoderm biopsy was performed from a 5th day blastocyst embryo by laser assisted hatching (LAH) to obtain 8 trophectoderm cells. In the specific operation, an Olympus IX71 microscope equipped with a Saturn laser system was used, and the laser power was set to 70%, 80% and 90% for comparative tests. The results showed that when the laser power was set to 80% and the pulse time was 1.0ms, an incision of appropriate size (about 6-8μm) could be formed in the trophectoderm of the blastocyst, which was convenient for accurately sucking 8 cells without damaging the inner cell mass. After obtaining the biopsy sample, the embryo was returned to the incubator for recovery for 2 hours, and its morphological recovery was observed. In this embodiment, 96% of the embryos were able to complete the re-expansion of the cystic cavity within 2 hours, indicating that the safety of the biopsy operation was good.

[0035] In this example, three sample processing methods were compared and studied: thermal lysis, alkaline lysis, and enzyme lysis. In the thermal lysis method, the sample was added with 20 μL of pure water and heated at 95°C for 10 minutes; the alkaline lysis method added 5 μL of 50 mM NaOH solution to the sample, treated at 95°C for 5 minutes, and then neutralized by adding 5 μL of 50 mM Tris-HCl (pH 8.0); the enzyme lysis method placed the sample in 20 μL of lysis buffer (10 mM Tris-HCl pH 8.0, 50 mM KCl, 1.5 mM MgCl 2 , 0.5% Tween-20, 100μg / ml proteinase K), incubated at 56°C for 30 minutes, and then inactivated at 95°C for 10 minutes. By comparing the amount and integrity of DNA released by the three methods, it was found that the enzymatic lysis method had the highest DNA release efficiency (92%) and the best DNA integrity, so the enzymatic lysis method was selected in this example. The lysed sample was divided into 3 equal parts, each of about 6.5μL, for subsequent detection. Without amplification, the lysis solution is used directly for detection, which can simplify the experimental process and reduce operational errors. This example shows that the amount of DNA released from 8 trophectoderm cells (about 48pg) is sufficient for subsequent detection without the need for additional DNA amplification steps.

[0036] In the present embodiment, LbCas12a protein is used, and the protein is derived from Lachnospiraceaebacterium, with high activity and fast reaction speed. A series of concentration gradient experiments (25nM, 50nM, 75nM, 100nM and 125nM) were carried out, and 75nM was determined to be the optimal working concentration, and the signal intensity was high and the background was low at this concentration. CrRNA design is the key link of the present embodiment, and is carried out based on the following principles: crRNA targeting 21q22.3 region for chromosome 21, which is almost constant in three copies in Down syndrome; Internal reference selection chromosome 4, because its aneuploid incidence is extremely low, almost constant diploid state is maintained. CrRNA sequence design ensures that GC content is 45-55%, avoids forming obvious secondary structure, and confirms that there is uniqueness in the whole genome.

[0037] Preferably, in this embodiment, the CRISPR-Cas12a reaction system (total volume 25 μL) comprises the following components:

[0038] LbCas12a protein: 75nM (final concentration)

[0039] Chromosome 21 crRNA (5'-GUACGAUUCGAGUGAUACCGUUUUAAUUUCUACUAAGUGUAGAU-3'): 100 nM;

[0040] Chromosome 4 crRNA (internal reference) (5′-GAUACUCGACGCUUAAGUAGGUUUUAAUUUCUACUAAGUGUAGAU-3′): 100 nM;

[0041] Fluorescent substrate (FAM-TTATT-BHQ1): 250 nM;

[0042] Sample DNA: 6.5 μL;

[0043] Reaction buffer (20 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl 2 , 1mM DTT, 5% glycerol): make up to 25μL;

[0044] In addition, we also tested different concentrations of fluorescent substrate (100nM, 150nM, 200nM, 250nM, 300nM and 350nM) and found that when the substrate concentration was 250nM, the signal intensity to background noise ratio was optimal. For the buffer composition, through orthogonal experiments, it was found that when the Tris-HCl concentration was 20mM, the KCl concentration was 100mM, and the MgCl 2 Cas12a has the highest activity at a concentration of 5mM. In addition, adding 1mM DTT and 5% glycerol can significantly improve the stability of Cas12a and prolong its activity retention time.

[0045] ;In this embodiment, detection was performed on a Bio-Rad CFX96 real-time fluorescence quantitative PCR instrument, the reaction temperature was set to 37 ° C constant temperature, the total reaction time was 60 minutes, and the fluorescence signal of the FAM channel (excitation / emission: 495nm / 520nm) was recorded every 3 minutes. We also studied the effect of different temperatures (25 ° C, 30 ° C, 35 ° C, 37 ° C, 40 ° C and 42 ° C) on the reaction efficiency and found that 37 ° C was the optimal temperature for Cas12a activity, at which the signal grew fastest, and the signal intensity that could clearly distinguish different chromosome copy numbers could be reached within 30 minutes.

[0046] like Figure 4 As shown, the chromosome copy number status is determined by calculating the ratio of the fluorescence signals of chromosome 21 and chromosome 4. The following judgment criteria are set for different copy number states:

[0047] Ratio range 0.8-1.2: judged as normal diploid;

[0048] Ratio range of 1.4-1.6: determined as trisomy 21;

[0049] Ratio range 0.4-0.6: judged as 21 monomers;

[0050] The above judgment criteria are determined based on theoretical expectations and statistical analysis of a large amount of experimental data. When the chromosome is normal diploid, the copy number ratio of the target and internal reference chromosomes is 1:1, so the signal ratio should be close to 1; when there is trisomy 21, the copy number ratio of the target and internal reference chromosomes is 3:2, and the theoretical ratio should be 1.5; when there is monosomy 21, the copy number ratio of the target and internal reference chromosomes is 1:2, and the theoretical ratio should be 0.5. In actual detection, considering the systematic error and biological variation, the judgment range will be appropriately relaxed and a buffer zone will be set. If the signal ratio falls in the buffer zone such as 0.65-0.75 or 1.3-1.35, it is recommended to repeat the test to confirm the result.

[0051] To verify the reliability of this method, we used 30 embryo samples with known karyotypes for blind testing, including 20 normal diploid samples, 8 trisomy 21 samples, and 2 monosomy 21 samples. The results showed that the consistency rate between this method and the gold standard NGS method was 96.7% (29 / 30). The only inconsistent sample was confirmed to be a mosaic after further analysis, containing about 20% trisomy cells, which was lower than the detection threshold of standard NGS, but was detected by this method, indicating that this method has a high sensitivity for low-proportion aneuploid mosaics.

[0052] In addition, by performing three repeated tests on 10 samples, the intra-batch coefficient of variation (CV) was calculated to be ≤5% and the inter-batch coefficient of variation was ≤10%, proving that this method has good repeatability and stability. From sample acquisition to result output, the entire testing process only takes about 2.5 hours, which is significantly shorter than the traditional NGS method (usually takes 24-48 hours), proving the huge advantage of this method in terms of detection speed.

[0053] This example demonstrates that the basic single-tube CRISPR-Cas12a detection system can quickly and accurately identify the copy number status of chromosome 21, and is suitable for rapid screening of clinical PGT-A, especially for clinical scenarios where results need to be obtained in a short time.

[0054] Example 2: Multicolor multiplex CRISPR-Cas12a aneuploidy detection

[0055] This embodiment provides a multicolor multiplex detection method based on the CRISPR-Cas12a system, which can simultaneously detect the aneuploidy status of multiple chromosomes in a single reaction system, significantly improving the detection efficiency.

[0056] In this example, 10 trophectoderm cells were obtained from blastocyst stage embryos at day 5-6. The biopsy method was similar to that in Example 1, but the laser power was optimized to 75%, and the pulse time was adjusted to 1.2 ms to form a 7-9 μm incision, which facilitated the acquisition of more cells to meet the needs of multiple detection. Unlike the direct lysis method used in Example 1, this example used an alkaline lysis method combined with a DNA amplification strategy to provide sufficient DNA for multiple detection. The specific steps are as follows:

[0057] First, the sample was placed in 5 μL of 50 mM NaOH solution and treated at 95°C for 5 minutes, after which 5 μL of 50 mM Tris-HCl (pH 8.0) was added for neutralization. Further, multiple displacement amplification (MDA) was performed using REPLI-g Single Cell Kit (QIAGEN). The reaction system contained phi29 DNA polymerase (1 U / μL), dNTPs (1 mM) and random hexamer primers (50 μM). The reaction conditions were 30°C and the amplification time was 2 hours. By comparing the yield and uniformity of different amplification times (1 hour, 2 hours, 3 hours and 4 hours), 2 hours was determined to be the optimal amplification time. Under this condition, the DNA yield was about 1.5 μg, and the amplification uniformity of different chromosome regions was good (CV≤15%). After amplification, 10 μL of the product was taken for subsequent detection.

[0058] In this embodiment, AsCas12a protein (derived from Acidaminococcus sp.) is used instead of LbCas12a because it has higher specificity and lower background activity, which is particularly suitable for multiple detection scenarios. The optimal working concentration was determined to be 100nM by gradient experiments (50nM, 75nM, 100nM, 125nM and 150nM), at which the signals of each channel were clearly distinguishable and did not interfere with each other.

[0059] The key to multiple detection lies in the rational allocation of crRNA design and fluorescent channels. In this embodiment, specific crRNA is designed for each target chromosome, and fluorescent reporter substrates of different wavelengths are provided to achieve multi-channel parallel detection. The crRNA design takes into account factors such as sequence specificity, GC content balance (42-58%), PAM sequence requirements, and non-interference in multiple systems. For example, the 21st chromosome crRNA targets the 21q22.3 region, and the 18th chromosome crRNA targets the 18q21.2 region. Each crRNA sequence is confirmed to be unique in the whole genome by bioinformatics analysis.

[0060] Preferably, in this embodiment, the multicolor multiplex CRISPR-Cas12a reaction system (total volume 50 μL) comprises the following components:

[0061] AsCas12a protein: 100 nM;

[0062] Each chromosome crRNA: 50nM each, including:

[0063] Chromosome 21 crRNA: Targeting the 21q22.3 region;

[0064] Chromosome 18 crRNA: Targeting the 18q21.2 region;

[0065] Chromosome 13 crRNA: Targeting the 13q14.2 region;

[0066] X chromosome crRNA: targeting the Xp11.23 region;

[0067] Y chromosome crRNA: Targeting the SRY gene region;

[0068] Chromosome 16 crRNA (internal reference): targeting the 16p13.3 region;

[0069] Fluorescent substrates (each corresponding to a different chromosome):

[0070] Chromosome 21: FAM-TTATT-BHQ1 (250 nM);

[0071] Chromosome 18: HEX-TTATT-BHQ1 (250 nM);

[0072] Chromosome 13: ROX-TTATT-BHQ2 (250 nM);

[0073] X chromosome: Cy5-TTATT-BHQ2 (300 nM);

[0074] Y chromosome: Cy5.5-TTATT-BHQ3(300nM);

[0075] Chromosome 16 (internal reference): TAMRA-TTATT-BHQ2 (250 nM);

[0076] Amplified sample DNA: 10 μL;

[0077] Reaction buffer (20 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl 2 , 1mM DTT, 5% glycerol): make up to 50μL;

[0078] Multiple candidate crRNAs were designed for each chromosome, and the combination with the highest signal specificity and the least interference between channels was screened through experiments. At the same time, the balance of signals in each channel was optimized by adjusting the concentration of different fluorescent substrates (within the range of 150-350nM). For example, the concentration of the fluorescent substrate for chromosomes X and Y was set to 300nM, which is slightly higher than that of other chromosomes (250nM), because the background noise of long-wavelength fluorophores is relatively high, and a higher concentration is required to increase the signal intensity.

[0079] In this example, the detection was performed on a Bio-Rad CFX96 real-time fluorescence quantitative PCR instrument equipped with a multi-channel detection function:

[0080] Temperature: 37°C constant temperature;

[0081] Duration: 80 minutes;

[0082] Detection mode: six channels: FAM, HEX, ROX, Cy5, Cy5.5 and TAMRA;

[0083] Acquisition frequency: record the fluorescence signal of each channel every 2 minutes;

[0084] The temperature gradient experiment (30-42°C) determined that 37°C was the optimal reaction temperature. The signal intensity changes at different reaction times (40, 60, 80, 100 and 120 minutes) were investigated, and 80 minutes was determined to be the optimal reaction time, at which time the signals of each channel reached a stable level and the interpretation window was the widest.

[0085] like Figure 5 As shown, the chromosome copy number status is determined by calculating the fluorescence signal ratio of each chromosome to the internal reference chromosome (No. 16):

[0086] Ratio range 0.8-1.2: judged as normal diploid;

[0087] Ratio range 1.4-1.6: determined as trisomy;

[0088] Ratio range 0.4-0.6: judged as monomer;

[0089] For chromosome Y: a signal significantly higher than the background (signal threshold ≥ 3 times the background) is considered positive (male), and no signal or a signal below the threshold is considered negative (female);

[0090] When interpreting the results, the slope data of 30-60 minutes is given priority, rather than the end-point fluorescence value, which can reduce the interference of nonspecific signals. In addition, an interpretation algorithm based on kinetic parameters has been developed, which comprehensively considers multiple parameters such as signal growth rate, signal threshold time and maximum signal intensity, thereby improving the reliability of interpretation.

[0091] To verify the reliability of the multi-color multiplex detection method, we used 50 embryo samples with known karyotypes for blind testing, including 30 normal karyotype samples, 12 various aneuploid samples (including trisomy 21, trisomy 18, trisomy 13, etc.) and 8 samples with abnormal sex chromosomes (such as 45,X, 47,XXY, etc.). The results showed that the overall consistency rate of this method with the gold standard NGS method was 94.0% (47 / 50). Among the three inconsistent samples, two were low-proportion mosaics (the mosaic ratio was about 15-25%), and one was a complex karyotype abnormality (microdeletions and trisomy of some chromosomes existed at the same time).

[0092] Further analysis showed that for simple aneuploidy samples, the detection accuracy of this method was as high as 97.6%; for mosaic samples, when the mosaic ratio was ≥30%, the detection accuracy was 91.7%; for low-proportion mosaics (mosaic ratio <30%), the detection accuracy dropped to 82.4%. This shows that this method has certain limitations for mosaic detection, but it is still better than most traditional methods.

[0093] The advantage of multi-color multiplex detection method is that it can detect multiple chromosomes at the same time, reducing sample consumption and detection time. Figure 5 As shown in the figure, the signals of different chromosomes can be clearly distinguished in different fluorescence channels, which is convenient for judging the status of multiple chromosomes at the same time. From sample acquisition to result output, the whole process takes about 3.5 hours, which is significantly faster than the traditional method, but slightly longer than the single-tube method, which is due to the extra time consumption of multi-channel data acquisition and analysis.

[0094] This example demonstrates that the multicolor multiplex CRISPR-Cas12a detection system is capable of simultaneously detecting the aneuploidy status of multiple chromosomes in a single reaction, significantly improving the detection efficiency, and is particularly suitable for application scenarios in conventional PGT-A screening that require simultaneous evaluation of the status of multiple chromosomes.

[0095] Example 3: Droplet digital CRISPR-Cas12a aneuploidy detection

[0096] This embodiment provides a high-precision chromosome aneuploidy detection method based on droplet digital CRISPR-Cas12a technology, which is particularly suitable for detecting subchromosomal level variations and low-proportion mosaics.

[0097] Different from the first two embodiments, the present embodiment uses the 3rd day cleavage stage embryo (8-12 cell stage) to perform blastomere biopsy, and obtains 2 blastomeres. The biopsy adopts laser assisted hatching technology, and the power is set to 65%, 70%, 75% and 80% for comparative test. The result shows that a suitable incision of 5-6 μm can be formed under 75% power, which is convenient to accurately absorb a single blastomere. After the biopsy, the embryo continues to be cultured to the blastocyst stage, and its development is observed. The result shows that 95% of the embryos can continue to develop to the blastocyst stage, indicating that the biopsy operation has less influence on embryo development.

[0098] In terms of sample processing, this embodiment uses a method of thermal lysis combined with whole genome amplification. The specific steps are as follows:

[0099] 1. Add 10 μL of pure water directly to the sample and heat it at 95°C for 10 minutes;

[0100] 2. Use MALBAC (Multiple Annealing and Looping Based Amplification Cycles) method for whole genome amplification:

[0101] Phase 1: 8 cycles of preamplification using MALBAC primers with a common sequence, with temperature cycles of 94°C for 20 seconds, 65°C for 30 seconds, and 72°C for 40 seconds;

[0102] Phase II: 18 cycles of PCR amplification using universal primers, with temperature cycles of 94°C for 20 seconds, 58°C for 30 seconds, and 72°C for 3 minutes;

[0103] 3. The amplified product was purified using AMPure XP magnetic beads (product: magnetic beads ratio was 1:1.8) and finally eluted with 15 μL TE buffer;

[0104] 4. Quantification using Qubit dsDNA HS Assay Kit, amplification product concentration range of 25-45ng / μL

[0105] The reason for choosing the MALBAC method instead of MDA is that MALBAC can provide more uniform genome coverage, especially in subchromosomal region detection, where uniformity of coverage is crucial. By comparing the amplification uniformity of different amplification methods (MALBAC, MDA, PicoPLEX), it was found that the coverage uniformity CV value of the MALBAC method within the 100kb interval was 18%, which was significantly better than MDA (CV value of 32%) and PicoPLEX (CV value of 25%).

[0106] In this embodiment, EnCas12a protein is used, which is an engineered Cas12a variant with higher activity and looser PAM requirements (capable of recognizing 5'-TTTV-3' and 5'-TTTT-3' sequences). Through concentration gradient experiments (30nM, 40nM, 50nM, 60nM and 70nM), 50nM is determined to be the optimal working concentration for droplet digital detection, and the signal intensity in a single droplet is most obvious compared with the background at this concentration.

[0107] Preferably, in this embodiment, the droplet digital CRISPR-Cas12a reaction system (total volume 20 μL, dispersed into droplets later) comprises the following components:

[0108] EnCas12a modified protein: 50nM;

[0109] Target chromosome region crRNA: 80nM;

[0110] Internal reference chromosome (1 and 4) crRNA: 40 nM each;

[0111] Fluorescent substrate (FAM-TTATT-BHQ1): 300nM;

[0112] Amplified sample DNA: 5ng;

[0113] Droplet formation reagent: according to the ratio recommended by Bio-Rad QX200 system;

[0114] Reaction buffer: make up to 20 μL;

[0115] In terms of crRNA design, considering the need to detect subchromosomal level variations, specific crRNAs were designed for different regions (13q14.2 and 13q14.3) of chromosome 13. 3-5 different crRNAs were designed for each region, covering a sequence range of about 100kb to improve the reliability of region-specific detection.

[0116] Droplet generation and detection using the Bio-Rad QX200 Droplet Digital PCR System:

[0117] 1. Mix 20 μL of the reaction system with 70 μL of droplet generation oil and use a QX200 droplet generator to generate about 20,000 uniform microdroplets (about 90 μm in diameter);

[0118] 2. Transfer the droplets to a 96-well plate, seal it and incubate it at 37°C for 90 minutes;

[0119] 3. Use QX200 droplet reader to detect the fluorescence intensity of each droplet, with the excitation light wavelength of 488nm and the emission light wavelength of 520nm;

[0120] 4. Divide the droplets into positive and negative according to the fluorescence intensity threshold (usually set to the negative control mean + 5 × standard deviation), and calculate the proportion of positive droplets;

[0121] The advantage of droplet digital technology lies in its absolute quantitative capability and single-molecule sensitivity. By dispersing the reaction system into thousands of microdroplets, each droplet contains 0-1 target DNA molecules, digital detection is achieved, which greatly improves the detection accuracy and is particularly suitable for detecting low-abundance variants. After testing, the detection limit of this system can reach 0.5%, which is much better than conventional CRISPR-Cas12a detection (about 5%) and NGS methods (about 20%).

[0122] like Figure 6 As shown, the chromosome copy number status is determined by calculating the ratio of the target chromosome region to the internal reference chromosome positive droplets:

[0123] Ratio range 0.85-1.15: judged as normal diploid;

[0124] Ratio range 1.40-1.60: judged as complete trisomy;

[0125] Ratio range 1.20-1.35: judged as about 30% mosaic trisomy;

[0126] Ratio range 0.60-0.80: judged as about 30% chimera monomer;

[0127] For the detection of subchromosomal variations, multiple crRNAs designed for specific regions were used to compare the signal intensity differences in different regions. Figure 6 As shown, by comparing the proportion of positive droplets in different regions of chromosome 13, microdeletions in the 13q14.2-q14.3 region can be detected.

[0128] To verify the reliability of the droplet digital CRISPR-Cas12a detection method, we used 25 samples for testing, including 10 normal samples, 8 known aneuploid samples, and 7 suspected chimera samples. The results showed that the aneuploidy detected by this method was 100% consistent with the NGS method; for chimera samples, the chimera ratio detected by this method was highly correlated with the NGS method (correlation coefficient r=0.92). More importantly, this method can detect chimera ratios as low as 10%, which is significantly better than conventional NGS methods (usually with a detection limit of 20%).

[0129] In terms of subchromosomal variation detection, this method successfully detected 3 known microdeletion / microduplication samples, including microdeletion in the 13q14.2-q14.3 region (about 2.5Mb), microdeletion in the 5p15.33 region (about 1.8Mb), and microduplication in the 22q11.21 region (about 3.0Mb). These results are completely consistent with the results of the microarray comparative genomic hybridization (aCGH) method, proving the reliability of this method in detecting subchromosomal variation.

[0130] The advantages of droplet digital CRISPR-Cas12a technology lie in its absolute quantitative capability and sensitivity to detect low-abundance variants. Figure 6 As shown in the figure, by counting the number of droplets, the chromosome ratio in the sample can be accurately calculated, which is particularly suitable for mosaic detection and subchromosome level variation analysis. From sample acquisition to result output, the entire process takes about 4 hours, which is slightly longer than the single-tube method and multi-color multiplex method, but has significant advantages in detection accuracy.

[0131] This example demonstrates that the droplet digital CRISPR-Cas12a detection system is capable of high-precision chromosome aneuploidy detection, and is particularly suitable for the detection of subchromosomal level variations and low-proportion chimeras, filling the gaps in conventional methods in this field.

[0132] Example 4: Time-resolved CRISPR-Cas12a aneuploidy detection

[0133] This embodiment provides a CRISPR-Cas12a aneuploidy detection method based on the time resolution principle, which utilizes the difference in activation rates of different crRNA-Cas12a complexes to distinguish different chromosome signals through a single fluorescence channel, reduces equipment requirements, and is suitable for detection applications in resource-limited environments.

[0134] The sample acquisition and processing methods in this embodiment are basically the same as those in Example 1. 5-8 trophectoderm cells are obtained from the 5th day blastocyst stage embryo, but a lower laser power (65%) and a shorter pulse time (0.8ms) are used to reduce potential damage to the embryo. For sample processing, a modified alkaline lysis method is used: the sample is added to 4μL 50mM NaOH solution, treated at 65°C for 3 minutes, then treated at 95°C for 2 minutes, and then 4μL 50mM Tris-HCl (pH 8.0) is added for neutralization. This method is milder than the standard alkaline lysis method, with better DNA integrity, but slightly lower efficiency (DNA release rate is about 85%). In the case of insufficient sample DNA, the incubation time can be increased to 5 minutes to increase the DNA release rate.

[0135] This embodiment uses FnCas12a protein (derived from Francisella novicida) with better thermal tolerance, which has a wide operating temperature range (35-48 ° C), suitable for use in resource-limited environments. The preferred working concentration is 120nM, and the signal growth rate is moderate at this concentration, which is convenient for distinguishing signal changes in different time windows.

[0136] The core innovation of this embodiment is to use the difference in activation rates of different crRNA-Cas12a complexes to achieve time-sharing detection of multiple chromosomes in a single fluorescence channel. Experiments have found that by carefully designing the crRNA sequence, the detection signals of different chromosomes can appear in different time windows, thereby achieving multiple detection without the need for a multi-channel fluorescence detector.

[0137] Preferably, in this embodiment, the time-resolved CRISPR-Cas12a reaction system (total volume 30 μL) comprises the following components:

[0138] FnCas12a protein: 120nM;

[0139] crRNA in each time window:

[0140] Chromosome 21 crRNA (fast window, 0-20 minutes): 100 nM;

[0141] Chromosome 18 crRNA (medium speed window, 20-40 minutes): 120nM;

[0142] Chromosome 13 crRNA (slow window, 40-60 minutes): 150nM;

[0143] Chromosome 4 crRNA (internal reference, 60-80 minutes): 180nM;

[0144] Universal fluorescent substrate (FAM-TTATT-BHQ1): 300nM;

[0145] Sample DNA: 8 μL;

[0146] Reaction buffer (20 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl 2 , 1mM DTT, 5% glycerol): make up to 30μL;

[0147] crRNA design is the key to time-resolved detection, and the activation rate needs to be controlled by adjusting the sequence characteristics. Through systematic research, it was found that the following factors affect the crRNA activation rate: ① GC content: the higher the GC content, the slower the activation rate; ② Sequence complexity: crRNA containing repeated sequences or single base regions has a faster activation rate; ③ Secondary structure: crRNA that forms a stable secondary structure has a slower activation rate. Based on these principles, we designed a series of crRNAs with different activation rates, tested their activation kinetics under the same conditions, and screened out the optimal combination that reached the peak in different time windows.

[0148] At the same time, by adjusting the concentration of different crRNAs (chromosome 21 crRNA uses a lower concentration of 100nM, and the internal reference crRNA uses a higher concentration of 180nM), the intensity balance of the signal in each time window is further optimized. The higher the crRNA concentration, the earlier it reaches the peak, but this is contrary to the influence of sequence characteristics and needs to be considered comprehensively.

[0149] In this embodiment, the reaction was carried out at 42 ° C constant temperature for a total of 80 minutes, and the FAM channel fluorescence signal was recorded every 1 minute. The reason for selecting a higher temperature (42 ° C instead of 37 ° C) is that FnCas12a is most active at this temperature, and high temperature helps to reduce nonspecific binding and improve the resolution of the time window.

[0150] The experiment showed that under the optimized conditions, the detection signals of the four chromosomes can be clearly distinguished in different time windows: the chromosome 21 signal reaches a peak at 10-15 minutes, the chromosome 18 signal reaches a peak at 30-35 minutes, the chromosome 13 signal reaches a peak at 50-55 minutes, and the internal reference chromosome 4 signal reaches a peak at 70-75 minutes. The windows between different chromosomes are well separated, and the signal overlap rate is <5%.

[0151] The data analysis of time-resolved detection is based on the signal change rate (dF / dt) rather than the absolute fluorescence value. The first-order derivative of the fluorescence curve is calculated to obtain the signal change rate curve at each time point, and the peak value of each time window is found and compared with the peak value of the internal reference window to determine the chromosome copy number status. The judgment criteria are as follows:

[0152] Target / reference peak ratio range 0.8-1.2: judged as normal diploid;

[0153] Target / reference peak ratio range 1.4-1.6: determined as trisomy;

[0154] Target / reference peak ratio 0.4-0.6 range: determined as monomer;

[0155] The peak appearance time also provides important information: if the peak appearance time is significantly advanced or delayed (>5 minutes), it may indicate the presence of sequence variation or complex aneuploidy in the sample, which requires further verification.

[0156] To verify the reliability of the time-resolved CRISPR-Cas12a detection method, we used 20 embryo samples with known karyotypes for testing, including 12 normal samples and 8 aneuploid samples. The results showed that the overall consistency between this method and the NGS method was 90.0% (18 / 20). Of the two inconsistent samples, one was a low-proportion mosaic and the other was a complex aneuploidy (multiple chromosome abnormalities at the same time), which reflects the limitations of time-resolved detection in complex samples.

[0157] Although the accuracy is slightly lower than that of multi-color multiplex detection, the biggest advantage of the time-resolved detection method is that it requires low equipment. Only a single-channel fluorescence detector is needed to complete the detection of multiple chromosomes, which greatly reduces the cost of detection. This makes this method particularly suitable for laboratories or field detection scenarios with limited resources. From sample acquisition to result output, the entire process takes about 3 hours, the operation steps are simple, and the equipment investment is only 20-30% of that of a multi-channel system.

[0158] This example demonstrates that the time-resolved CRISPR-Cas12a detection system can detect aneuploidy of multiple chromosomes under single-channel conditions, providing an economical and practical solution for PGT-A detection in resource-limited environments.

[0159] Example 5: Non-invasive CRISPR-Cas12a embryo culture fluid detection system

[0160] This embodiment provides a non-invasive aneuploidy detection method based on free DNA in embryo culture medium, which does not require embryo biopsy and does not interfere with embryo development at all, and provides a new non-invasive solution for PGT-A technology.

[0161] Different from the previous four embodiments, this embodiment does not perform embryo biopsy, but uses the free DNA naturally released into the culture medium during the in vitro culture of the embryo for detection. The specific operation is as follows:

[0162] 1. Embryo culture adopts the standard single embryo culture mode, each embryo is placed in a microdroplet containing 15-20μL culture solution, and the outer layer is covered with light mineral oil to prevent evaporation;

[0163] 2. Starting from the 3rd day of embryo culture, replace the culture medium with fresh one every 24 hours;

[0164] 3. On day 5 (blastocyst stage), collect 10-20 μL of culture medium and place it in a microtube containing 1 μL of DNA protection reagent (containing 10 mM EDTA and 0.1% SDS);

[0165] 4. Samples can be tested immediately or stored at -80°C;

[0166] By comparing the DNA content in the culture medium at different stages, it was found that the free DNA content in the culture medium of the blastocyst stage on day 5 was the highest, with an average of 3-8pg. At the same time, the method of directly collecting the culture medium and collecting PBS after rinsing the blastocyst with PBS was compared. The results showed that the DNA yield of directly collecting the culture medium was higher and the quality was better.

[0167] Since the free DNA content in the culture medium is extremely low, usually below the minimum requirement for conventional PCR and CRISPR-Cas12a detection, special DNA enrichment treatment is required. This example uses an optimized DNA magnetic bead enrichment system:

[0168] 1. Add 3 volumes of homemade binding buffer (20% PEG8000, 2.5M NaCl, 10mM Tris-HCl pH 8.0) to the culture fluid sample (10-20μL);

[0169] 2. Add 1.8 times the sample volume of homemade silica magnetic bead suspension (50 mg / mL silica magnetic beads);

[0170] 3. Incubate at room temperature for 15 minutes, mixing gently every 3 minutes;

[0171] 4. Magnetic separation and washing twice (using 70% ethanol);

[0172] 5. Elute the DNA using 15 μL of elution buffer (10 mM Tris-HCl pH 8.0) preheated to 65°C.

[0173] By comparing the efficiency of different enrichment methods (commercial kits, traditional phenol-chloroform extraction, manual magnetic bead enrichment), it was found that the optimized magnetic bead enrichment system had the highest recovery rate (≥85%) and the lowest loss rate (≤15%) in ultra-trace DNA (≤10pg) samples. In addition, the recovery efficiency of extremely trace DNA can be further improved by adding carrier RNA (10ng / μL).

[0174] Since the free DNA content in the culture medium is extremely low and may be fragmented, an enhanced CRISPR-Cas12a detection system is needed to improve sensitivity. This example uses highly active EnCas12a protein and enhances detection sensitivity through a variety of strategies:

[0175] Preferably, in this embodiment, the enhanced CRISPR-Cas12a reaction system (total volume 25 μL) comprises the following components:

[0176] Highly active EnCas12a protein: 60nM;

[0177] Optimized chromosome-specific crRNA mixture: 120nM;

[0178] Enhanced fluorescence substrate (FAM-TTATTATTAT-BHQ1): 400nM;

[0179] Enriched DNA: 10 μL;

[0180] Reaction buffer (optimized ionic strength): make up to 25 μL;

[0181] Several key innovations of this embodiment include:

[0182] 1. Use the improved EnCas12a protein, which is about 3 times more active than the standard LbCas12a and can be quickly activated under low DNA concentration conditions;

[0183] 2. Use an extended fluorescent substrate (TTATTATTAT instead of the standard TTATT) to increase the chance of binding to the activated Cas12a and improve the cutting efficiency;

[0184] 3. Design multiple crRNAs (3-5) for each target region and use them together to increase the coverage and recognition chance of the target sequence;

[0185] 4. Optimize the reaction buffer ionic strength and pH by reducing the KCl concentration to 80 mM and increasing the MgCl 2 The concentration was increased to 6 mM to improve the cleavage activity of Cas12a under low DNA concentration conditions;

[0186] After testing, the enhanced system has a detection limit of about 500 fg for DNA, which is about 20 times higher than the standard system (about 10 pg), meeting the needs of culture fluid sample testing.

[0187] In this embodiment, the reaction was carried out at 37°C for a total of 120 minutes, and the fluorescence signal was recorded every 2 minutes. The longer reaction time is to ensure that the signal of the extremely small amount of DNA sample can be fully accumulated to reach a reliable detection level.

[0188] Data analysis uses an optimized signal processing algorithm, including background correction, noise filtering and signal enhancement steps, to improve the detection rate of weak signals. The judgment criteria are basically the same as those in the above embodiment, but considering the particularity of the culture fluid sample, a more stringent quality control standard is set: the internal reference signal must reach more than 5 times the negative control within 90 minutes, otherwise it is judged as invalid detection.

[0189] A unique advantage of non-invasive testing is that it can be combined with embryo morphology scoring to conduct a multi-dimensional embryo quality assessment. This example establishes a comprehensive scoring system that combines the risk of chromosomal aneuploidy and the Gardner blastocyst scoring system to make a more comprehensive prediction of embryo development potential. Studies have found that this type of comprehensive assessment can more accurately predict the developmental potential of embryos after implantation than simple morphological scoring or simple chromosome testing, and the implantation success rate is increased by about 15%.

[0190] To verify the reliability of the non-invasive detection method, we used 40 pairs of samples (each pair included an embryo biopsy sample and a corresponding culture medium sample) for parallel validation. The results showed that the overall consistency between the non-invasive detection method and the biopsy detection method was 85.0% (34 / 40). Further analysis showed that the detection rate for euploid embryos was 92.3% (24 / 26), and the detection rate for aneuploid embryos was 71.4% (10 / 14). This indicates that the non-invasive detection rate for aneuploidy is high, which may be related to the pattern of DNA release from aneuploid embryos.

[0191] Discordant samples mainly appeared in chromosomal mosaic embryos, which may reflect the different sensitivities of different detection methods for embryonic heterogeneity. Interestingly, in 2 samples that were aneuploid in culture medium but normal in biopsy, subsequent whole embryo analysis confirmed the presence of a low proportion of aneuploid cells, indicating that culture medium detection may in some cases reflect embryonic heterogeneity that was not detected by biopsy.

[0192] The biggest advantage of non-invasive detection methods is that they do not interfere with embryonic development at all, avoiding the 5-10% risk of embryo damage that may be caused by biopsy, and are particularly suitable for cases with only a small number of available embryos. From collecting the culture medium to outputting the results, the entire process takes about 4.5 hours, which is slightly longer than the biopsy method, but considering its non-invasive nature, this time cost is acceptable.

[0193] This example demonstrates that the culture medium-based noninvasive CRISPR-Cas12a detection system provides a new noninvasive option for PGT-A. Although its detection accuracy is slightly lower than that of traditional biopsy methods, its noninvasive nature and zero interference with embryonic development make it uniquely valuable in specific clinical scenarios.

[0194] Example 6: Portable CRISPR-Cas12a aneuploidy detection system

[0195] This embodiment provides a portable CRISPR-Cas12a aneuploidy detection system suitable for resource-limited environments, which realizes rapid on-site detection by integrating simplified sample processing, stable reagent formulation and portable detection equipment, and is particularly suitable for use in primary medical institutions.

[0196] The portable detection system developed in this embodiment includes the following main components:

[0197] 1. Portable thermostat: weight about 500g, size 12×8×5cm, built-in lithium battery can work continuously for 8 hours, temperature control accuracy ±1°C, operating temperature range 25-95°C;

[0198] 2. Simple fluorescence detection equipment: based on blue light LED (wavelength 488nm) excitation and smartphone camera detection with a specific filter (wavelength 520nm), the sample and light path are fixed by a 3D printed bracket;

[0199] 3. Premixed lyophilized reagent: lyophilized beads containing all reaction components, with a shelf life of ≥6 months at room temperature and ≥12 months at 4°C;

[0200] 4. Smartphone APP: used for image acquisition, signal analysis and result interpretation, supporting Android and iOS systems;

[0201] The entire system weighs less than 1kg and can be packed into a portable box, making it suitable for use in the field or in resource-limited environments. The system cost is about 10-15% of standard laboratory equipment, greatly reducing the technical threshold and funding requirements.

[0202] This embodiment simplifies the sample processing process. After obtaining 5-8 cells from the blastocyst trophectoderm, the premixed lysis buffer (containing proteinase K) is directly added, incubated at 56°C for 20 minutes in a portable thermostat, inactivated at 95°C for 5 minutes, and directly entered into the detection system without DNA extraction or purification. This simplified process reduces the operation steps and the risk of cross-contamination, and is particularly suitable for non-professionals.

[0203] A key innovation of this embodiment is the development of a stable frozen premix reagent, which overcomes the reliance of conventional CRISPR-Cas12a systems on cold chain transportation and storage, making it more suitable for use in resource-limited environments. The composition of the frozen premix reagent is as follows:

[0204] Stable LbCas12a variant: 85nM (concentration after reconstitution);

[0205] Chromosomes 21, 18, 13 and internal reference crRNA: 75 nM each (concentration after reconstitution);

[0206] Fluorescent substrate (enhanced stability formula): 350nM (concentration after reconstitution);

[0207] Stabilizer mixture: contains sucrose (5%), trehalose (3%) and BSA (0.1%);

[0208] Reaction buffer components (lyophilized state);

[0209] When using, just add 20μL of pure water and 10μL of sample lysate to perform the test. By adding a specific stabilizer combination, the freeze-dried reagent can remain stable for ≥7 days in an environment up to 45°C and remain active for ≥6 months at room temperature (25°C). In contrast, traditional liquid reagents can only maintain activity for about 2 days at room temperature.

[0210] Stable LbCas12a variants were obtained by site-directed mutagenesis (T657R, S701R, and Q713R), which enhanced the stability of the protein in a dry state and under high temperature conditions, but had little effect on its catalytic activity (the activity remained about 90%).

[0211] The reaction in this example was incubated at 37°C in a portable thermostat for 60 minutes, and fluorescence was excited by a blue LED every 5 minutes and images were taken with a smartphone camera. The smartphone APP extracted fluorescence intensity information through an image analysis algorithm, calculated the signal change curve, and achieved semi-quantitative detection.

[0212] Compared with professional fluorescence detectors, this simplified system has a slightly lower sensitivity (the detection limit is about 20pg DNA, while the professional equipment is 10pg), but by extending the reaction time (60 minutes) and optimizing the image analysis algorithm, it can achieve a detection accuracy of nearly 90%. In addition, by separating the RGB channels of the mobile phone camera, simple two-color detection (FAM and ROX) can be achieved, further improving the detection throughput.

[0213] To verify the reliability of the portable CRISPR-Cas12a detection system, we conducted validation tests in five IVF centers of different sizes, testing a total of 100 embryo samples. The results showed that the overall consistency between the portable system and conventional laboratory methods was 91.0% (91 / 100). The inconsistent samples were mainly concentrated in the boundary value area (ratios close to 0.75 or 1.3), indicating that the system has limited resolution in these areas.

[0214] From sample acquisition to result output, the entire process takes an average of 2.8 hours, the system has good reproducibility, and the CV value between different operators is <15%. In a user experience survey, 90% of operators thought the system was "easy to use" or "very easy to use", and 85% thought the operating instructions were "clear" or "very clear". After a simple 1-hour training, personnel without a molecular biology background can also operate the system correctly and obtain reliable results.

[0215] The advantages of the portable system are its simplicity and applicability. It can be used in resource-limited environments without complex equipment, and is particularly suitable for use in primary care institutions. The system cost is only 10-15% of traditional equipment, and the cost per sample test is reduced by about 70%, which makes PGT-A technology accessible to a wider range of people.

[0216] This example demonstrates that the portable CRISPR-Cas12a aneuploidy detection system can achieve reliable chromosome aneuploidy detection in resource-limited environments, providing an economical and practical solution for the popularization of PGT-A technology.

[0217] Example 7: Multi-omics combined with CRISPR-Cas12a detection system

[0218] This example provides a multi-omics analysis method combining single-cell RNA sequencing (scRNA-seq) and CRISPR-Cas12a detection, which not only evaluates the chromosome copy number status, but also analyzes the gene expression profile, providing multidimensional information for the comprehensive evaluation of embryos.

[0219] In this example, 10-12 trophectoderm cells were obtained from the 5th day blastocysts, and then the cells were divided into:

[0220] 1. Place the cells in PBS containing 0.1% BSA and gently pipette to prepare a single-cell suspension;

[0221] 2. Use a micropipette to divide the single-cell suspension into two parts: 2-3 cells for RNA analysis and 7-9 cells for DNA analysis;

[0222] 3. Cell counting uses trypan blue staining to confirm cell number and activity (activity required to be ≥90%);

[0223] This triage strategy allows simultaneous acquisition of transcriptome and chromosome copy number information from the same embryo sample, enabling multidimensional analysis.

[0224] The RNA analysis process was as follows:

[0225] 1. Cell lysis and mRNA capture using a modified oligo(dT) magnetic bead method with a capture efficiency of ≥85%;

[0226] 2. Reverse transcription uses the SMART-Seq2 protocol and adds a unique molecular identifier (UMI);

[0227] 3. cDNA amplification uses 15-18 cycles of PCR reaction, with a yield of about 1-2ng;

[0228] 4. Use Nextera XT kit to construct sequencing library;

[0229] 5. Sequencing on the Illumina platform generates approximately 1 million reads per cell;

[0230] The single-cell RNA-seq data analysis focuses on the following aspects: ① expression levels of genes related to embryonic development; ② gene expression characteristics related to chromosomal aneuploidy; ③ gene sets related to embryonic development potential prediction; ④ changes in expression levels in specific chromosomal regions, which are used to verify CRISPR-Cas12a detection results.

[0231] The DNA analysis part uses an optimized CRISPR-Cas12a detection system, and the basic process is similar to Example 2, which realizes the parallel detection of the main aneuploidy risk chromosomes (21, 18, 13, X, Y). An innovative point of this example is the development of a DNA recovery method specifically for single-cell RNA-seq residues:

[0232] 1. Collect discarded supernatant (containing cellular DNA) from single-cell RNA-seq sample processing;

[0233] 2. Recover DNA by a modified DNA precipitation method (add 3 volumes of anhydrous ethanol and 0.1 volumes of 3 M sodium acetate, incubate at -20°C for 2 hours);

[0234] 3. Centrifuge at 15,000 g for 15 minutes, discard the supernatant, and wash once with 70% ethanol;

[0235] 4. Wash once with anhydrous ethanol, air-dry for 2-3 minutes, and dissolve in TE buffer;

[0236] This method can recover about 40-60% of cellular DNA from RNA-seq sample processing waste. Although the recovery rate is not high, combined with the high sensitivity of the CRISPR-Cas12a system, it is sufficient for chromosome copy number analysis. This "waste utilization" strategy maximizes the information output of limited samples and is particularly suitable for scenarios with extremely limited sample sizes, such as embryo biopsy.

[0237] The core value of this example is to integrate RNA-seq transcriptome data with CRISPR-Cas12a chromosome copy number data and develop a comprehensive scoring system that not only evaluates whether the chromosomes are normal, but also analyzes whether the gene expression pattern supports healthy embryonic development. The main scoring dimensions include:

[0238] 1. Chromosome copy number status: based on CRISPR-Cas12a detection results

[0239] 2. Effects of chromosomal aneuploidy on gene expression: Analyze whether gene expression levels in specific chromosomal regions are consistent with copy number abnormalities

[0240] 3. Expression of developmental potential-related gene sets: including cell cycle regulation, implantation-related and metabolism-related gene sets

[0241] 4. Embryonic development stage assessment: Determine whether the development stage is consistent with the morphological score based on the expression level of specific marker genes

[0242] These factors are integrated through a machine learning algorithm (random forest model) to generate a comprehensive score and implantation recommendation. The model training used 150 historical sample data with known implantation results, and the accuracy of the validation set reached 82%, which is significantly better than the prediction based solely on chromosome copy number (accuracy of about 70%) or morphological score (accuracy of about 65%).

[0243] To verify the reliability of the multi-omics combined detection system, we analyzed 30 blastocyst samples, and each sample was simultaneously subjected to conventional PGT-A testing, single-cell RNA-seq, and multi-omics analysis of this method. The results showed that in terms of chromosome copy number judgment, the consistency rate between this method and conventional PGT-A was 93.3% (28 / 30). More importantly, in the subsequent clinical follow-up, the implantation rate of embryos selected based on this method after implantation was 72.2% (13 / 18), which was significantly higher than the control group selected based only on PGT-A results (implantation rate 58.3%, 7 / 12), indicating that the additional information provided by multi-omics analysis does help improve the accuracy of embryo selection.

[0244] From sample acquisition to result output, the multi-omics analysis method takes about 12 hours, of which the CRISPR-Cas12a detection part takes about 3 hours and the RNA-seq part (including sequencing) takes about 9 hours. Although the total time is long, it is still significantly faster than the traditional combination method (usually takes more than 48 hours). In terms of cost, the cost of analyzing each sample is about 2,000 yuan, which is higher than the simple CRISPR-Cas12a detection (about 300 yuan), but lower than the traditional PGT-A+RNA-seq combination (about 3,500 yuan).

[0245] This example demonstrates that the multi-omics combined with CRISPR-Cas12a detection system can provide more comprehensive embryo assessment information than simple chromosome detection, which helps to improve the accuracy of embryo selection and the success rate of assisted reproductive technology. Although the cost and time are increased compared to simple CRISPR-Cas12a detection, considering its potential value in improving implantation rate, it still has a good cost-effectiveness ratio in specific clinical scenarios.

[0246] The above embodiments describe in detail the different implementations and application scenarios of the present invention, from basic single-tube detection to multi-omics joint analysis, covering a variety of technical solutions from simple, economical, and rapid conventional detection to high-precision, multi-dimensional comprehensive analysis. These embodiments fully demonstrate the flexibility and broad application prospects of the rapid detection technology of preimplantation chromosome aneuploidy based on the CRISPR-Cas12a system, and provide a series of new technical options for clinical PGT-A.

[0247] Although the above embodiments have described in detail various implementations of the present invention, those skilled in the art should understand that various deformation, modification and improvement may be made to these embodiments without departing from the spirit and scope of the technical solution of the present invention, and these deformation, modification and improvement should also be regarded as the scope of protection of the present invention.

Claims

1. A rapid detection method for preimplantation chromosome aneuploidy based on CRISPR-Cas12a, characterized in that: The method comprises the following steps: a) Obtaining a biopsy cell sample from the embryo; b) processing the biopsy cell sample to release genomic DNA; c) preparing a CRISPR-Cas12a detection reaction system, the reaction system comprising a Cas12a protein, a crRNA for a chromosome to be detected, a crRNA for an internal reference chromosome, and a fluorescent reporter substrate; d) adding the treated sample DNA to the CRISPR-Cas12a detection reaction system and reacting under appropriate conditions; e) detecting and recording changes in fluorescence signals; f) calculating the fluorescence signal ratio of the chromosome to be detected and the internal reference chromosome; and g) determining the copy number status of the chromosome to be detected according to the signal ratio.

2. The method according to claim 1, characterized in that The biopsy cell sample processing method in step b) is selected from one or more of the following: Thermal lysis method, the sample was added to purified water and heated at 95°C for 10 min; Alkaline lysis method: add NaOH solution to the sample, treat at 95°C for 3-5 minutes, and then add an equal volume of Tris-HCl to neutralize; For enzymatic lysis, samples were added to lysis buffer containing proteinase K, incubated at 56°C for 30 min, and then inactivated at 95°C for 10 min.

3. The method according to claim 1, characterized in that The step b) further comprises a step of amplifying the released genomic DNA, wherein the amplification method is selected from one or more of the following: Multiple displacement amplification (MDA); PicoPLEX / SurePlex technology; Multiple annealing loop ligation PCR-based amplification (MALBAC); or Direct detection method without amplification.

4. The method according to claim 1, characterized in that: The Cas12a protein is selected from one or more of the following: LbCas12a, working concentration 50-100 nM; AsCas12a, working concentration 75-125nM; FnCas12a, working concentration 80-150nM; or EnCas12a, working concentration is 40-80nM.

5. The method according to claim 1, characterized in that The design of the crRNA has the following characteristics: 19-25 nucleotides in length; GC content is 40-60%; The target sequence is adjacent to a PAM sequence having 5′-TTTV-3′, where V is A, C, or G; Unique across the genome; and 3-5 different crRNAs are designed for each chromosome to be detected, with a target spacing of 100kb-1Mb.

6. The method according to claim 1, characterized in that The fluorescent reporter substrate is selected from one or more of the following: Single-stranded DNA fluorescent substrate, the structure is 5'-fluorescent group-TTATT-quencher group-3', the concentration is 100-500nM; Molecular beacon-type substrates form a hairpin structure with lower background fluorescence; Polymer-supported substrates, where the substrate is coupled to the surface of microspheres or hydrogels; or Dual fluorophore substrates, with one fluorophore labeled at each end, produce FRET changes after cleavage.

7. The method according to claim 1, characterized in that Steps c) to e) use one or more of the following detection technology strategies: The split tube detection method uses one reaction tube to detect one chromosome; Multicolor detection method, using different fluorescent markers to detect multiple chromosomes in the same reaction tube; Time-resolved detection method, using the difference in activation rate of different crRNA-Cas12a complexes to distinguish different chromosome signals; or The droplet digital CRISPR detection method disperses the reaction system into thousands of microdroplets and calculates the copy number by counting the proportion of positive droplets.

8. The method according to claim 1, characterized in that The signal detection in step e) adopts one or more of the following methods: End point detection method, the fluorescence signal is detected once after the reaction is completed; Real-time kinetic detection method, continuously monitoring the changes in fluorescence signal during the reaction; or High-resolution detection method, high-frequency signal acquisition and data smoothing.

9. The method according to claim 1, characterized in that: The data analysis and result interpretation in steps f) and g) are performed by one or more of the following methods: Ratio analysis method, based on the ratio of the target chromosome signal to the internal reference chromosome signal, where a ratio of 0.4-0.6 is considered monosomy, 0.8-1.2 is considered normal diploid, and 1.4-1.6 is considered trisomy; Kinetic parameter analysis method, based on comprehensive judgment of parameters such as signal growth rate, signal threshold time and maximum signal intensity; or Machine learning-assisted analysis method performs comprehensive analysis based on fluorescence signal curve characteristics and multi-channel signal correlation.

10. The method according to claim 1, characterized in that The method is applied to one or more of the following scenarios: Preimplantation genetic testing (PGT-A); Non-invasive embryo testing uses free DNA in embryo culture medium for testing; Subchromosome level variation detection, including detection of microdeletions and microduplications; Testing for chromosome structural abnormalities; or Comprehensive assessment of embryos combined with single-cell RNA-seq.

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