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CRISPR-Cas system for diagnosing spinal muscular atrophy and application thereof

A technology for spinal muscular atrophy and diagnostic reagent, applied in the field of CRISPR-Cas system for the diagnosis of spinal muscular atrophy

Active Publication Date: 2020-10-23
SHANGHAI PINPOINT MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the application of CRISPR / Cas14a1 molecular detection technology in the diagnosis of genetic diseases in the world

Method used

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  • CRISPR-Cas system for diagnosing spinal muscular atrophy and application thereof
  • CRISPR-Cas system for diagnosing spinal muscular atrophy and application thereof
  • CRISPR-Cas system for diagnosing spinal muscular atrophy and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Cas14a1 protein expression and purification

[0063] The Cas14a1 expression vector was constructed and Cas14a1 protein was expressed in Escherichia coli, purified by affinity chromatography, and Cas14a1 protein was detected by western blotting.

[0064] 1) Cas14a1 protein expression

[0065] ①Transform the constructed plasmid containing the Cas14a1 gene into BL21(DE3) competent cells, evenly spread it on the LB plate containing 50 μg / mL kanamycin sulfate, and place it upside down in a 37°C incubator for overnight culture. Pick a single clone colony from the transformed plate and inoculate it into 4 mL of liquid LB medium containing 50 μg / mL kanamycin sulfate;

[0066] ②To be cultured to OD 600 0.5-0.8, add IPTG with a final concentration of 0.2mM to the LB culture medium, and then place them at 15°C and 37°C for 16 hours to induce expression;

[0067] ③ Centrifuge the induced culture medium at 12,000 rpm for 5 minutes, remove the supernatant, add PBS to resuspend the...

Embodiment 2

[0073] Detect the genotyped DNA and verify the method

[0074] Using normal human gDNA as a template, CRISPR / Cas14a1 cleavage detection was carried out after PCR amplification. By comparing the fluorescence increase values ​​produced by different buffers, the optimal buffer was Cutsmart buffer of NEB (recipe: 50mM KAc, 20mM Tris-Ac, 10mM Mg (Ac)2, 100 μg / mL BSA (pH7.9@25°C)). In the subsequent detection process, we all choose Cutsmart buffer as the buffer.

[0075] 1. crRNA transcription

[0076] (1) Design and synthesize the corresponding DNA sequence of crRNA as shown in the above table, and use HiScribe in vitro TM T7High Yield RNA Synthesis Kit (NEW ENGLAND BioLabs, E2040s) transcription kit was used for transcription to obtain crRNA.

[0077] The transcription system is as follows:

[0078]

[0079] Incubate at 37°C for 16 hours.

[0080] (2) Remove possible residual DNA in the transcription system by DNaseI

[0081]

[0082] Incubate at 37°C for 30 minutes. ...

Embodiment 3

[0100] Comparing the impact of FQ-probe of different lengths

[0101] We constructed FQ-probes with a length of 12nt and 21nt, and used the normal human gDNA amplification product as a template to perform CRISPR / Cas14a1 cleavage fluorescence detection, and the specific steps were similar to Example 2. The results showed that the Δ fluorescence value produced by the 21nt FQ-probe was significantly higher than that produced by the 12nt FQ-probe ( Figure 4 ), so we use a 21nt length FQ-probe.

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PUM

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Abstract

The invention relates to a CRISPR-Cas system, in particular to a CRISPR-Cas system for diagnosing spinal muscular atrophy and application thereof. The CRISPR-Cas system for diagnosing spinal muscularatrophy disclosed by the invention comprises Cas14a1, crRNA, ssDNA and FQ-probe. A method based on combination of CRISPR / Cas14a1 and asymmetric PCR constructed by the invention can perform gene detection for SMA patients, and can be used for specifically detecting the mutation condition of SMN1 gene. Different from an existing CRISPR / Cas12a SMA diagnosis technology, the kit can also distinguish SMN2 gene interference so as to judge whether a detected person suffers from the spinal muscular atrophy or not, and has the characteristics of simple and convenient operation, high specificity and lowcost.

Description

technical field [0001] The invention relates to a CRISPR-Cas system, in particular to a CRISPR-Cas system for diagnosing spinal muscular atrophy and its application. Background technique [0002] SMA is a neuromuscular disease with symmetrical muscle weakness and muscle atrophy caused by the degeneration of motor neurons in the anterior horn of the spinal cord. It is one of the most common autosomal recessive genetic diseases in infancy, mainly manifested in the proximal limbs. Muscle weakness, with the aggravation of the disease, the decline or loss of body motor function, swallowing and spontaneous breathing difficulties, and eventually death due to respiratory muscle paralysis. The incidence of SMA in the population is about 1 / 6000-1 / 10000 [1] . SMA is usually divided into three subtypes according to the severity of the disease and the age of onset, of which SMA-I accounts for about 50%. Patients develop symptoms at birth or within 6 months of birth. Died within two ye...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2531/107C12Q2521/327C12Q2525/161C12Q2563/107Y02A50/30
Inventor 梁德生胡志青周妙金邬玲仟
Owner SHANGHAI PINPOINT MEDICAL TECH CO LTD
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