Method capable of carrying out in-vitro off-target detection and sgRNA screening in batches

An off-target, batch technology, applied in the field of genetic engineering, to achieve the effect of high throughput, reliable experimental results and wide applicability

Active Publication Date: 2020-11-24
杭州舒桐生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] The purpose of the present invention is to solve the problem that the existing sgRNA off-target detection method can only detect whether one sgRNA is off-target each time, and how to screen sgRNAs with high cutting efficiency and low off-target, and provide a method that can perform in vitro off-target detection and sgRNA screening in batches Methods

Method used

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  • Method capable of carrying out in-vitro off-target detection and sgRNA screening in batches
  • Method capable of carrying out in-vitro off-target detection and sgRNA screening in batches
  • Method capable of carrying out in-vitro off-target detection and sgRNA screening in batches

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specific Embodiment approach 1

[0033] Specific Embodiment 1: In this embodiment, a method for in vitro off-target detection and sgRNA screening can be carried out in batches, which is completed according to the following steps:

[0034] 1. Collect 4×10 6 ~8×10 6 transfer the cells to a centrifuge tube, centrifuge, discard the medium, wash once with PBS, centrifuge again, discard the supernatant, and extract the cellular genomic DNA of the solid phase after centrifugation;

[0035] 2. Fragment the genomic DNA extracted in step 1 into a 300bp-700bp fragment, and then purify it with DNA purification magnetic beads;

[0036] 3. Perform end repair, add A tail, and add stem-loop structure linker 1 to the DNA purified in step 2, then treat it with exonuclease, and then treat it with ddNTP;

[0037] 4. Design and synthesize sgRNA oligo library, perform PCR amplification and in vitro transcription to obtain sgRNA pool;

[0038] 5. Use the Cas enzyme and the sgRNA pool obtained in step 4 to cut the ddNTP-treated D...

specific Embodiment approach 2

[0043] Specific embodiment 2: The difference between this embodiment and specific embodiment 1 is: in step 2, the instrument Bioruptor is used to interrupt DNA, parameters: DNA 50ng / μL; volume 100 μL; 15sON-90sOFF; 6-8 cycles; interruption The final DNA was purified with DNA purification magnetic beads, and the elution volume was 37 μL.

[0044] Other steps are the same as in the first embodiment.

specific Embodiment approach 3

[0045] Embodiment 3: The difference between this embodiment and Embodiment 1 or 2 is that in step 3, a kit is used to repair DNA ends, add A tails, and add stem-loop structure adapter 1, wherein the DNA ends are repaired, A tails are added Concrete reaction system and reaction procedure are as follows:

[0046] Kit: ABclonal Rapid DNA Library Construction Kit

[0047]

[0048] Reaction procedure: 20°C for 30 minutes; 65°C for 30 minutes to obtain the end repair mixture;

[0049] The specific reaction system and reaction procedure of adding stem-loop structure linker 1 are as follows:

[0050]

[0051] Reaction program: 1h at 22°C, then 1×DNA purification magnetic beads purification, elution volume 30 μL.

[0052] Other steps are the same as those in Embodiment 1 or 2.

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Abstract

The invention discloses a method capable of performing in-vitro off-target detection and sgRNA screening in batches, belonging to the technical field of gene engineering. The invention aims to solve the problem that an existing sgRNA off-target detection method can only detect whether one sgRNA is off-target or not every time and problems in screening of sgRNA with high cutting efficiency and lowoff-target rate. According to the high-throughput off-target detection method for sgRNA in the invention, in-vitro transcription is carried out in an sgRNA pool form, and the on-target or off-target condition of thousands of sgRNAs can be detected at the same time; and cutting efficiency is judged according to the number of on-target reads, and then, the off-target effect is judged according to off-target sites and the number of off-target reads. The method provided by the invention can realize in-vitro off-target detection and sgRNA screening in batches.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method capable of performing in vitro off-target detection and sgRNA screening in batches. Background technique [0002] The CRISPR-Cas9 system is derived from the acquired immune system of bacteria and archaea, and has become a powerful gene editing tool due to its simple and efficient operation. Since the discovery of CRISPR / Cas9 in early 2013 to achieve precise DNA editing in cells, it has shown a blowout development in recent years. Compared with gene editing technologies such as ZFN and TALEN, it is simpler, more economical and easier to operate, making it the most effective, cheap and easy gene editing method so far. point mutation), gene therapy (sickle cell anemia HBB gene repair, etc.), live cell imaging (such as Cas-FISH), functional gene screening (CRISPRi / a positive and negative screening), targeted capture (CRISPR-capture), etc. It has very important ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013C12N15/1089C12N15/1093
Inventor 程欢欢陈莹谢红娴兰凯黄龙
Owner 杭州舒桐生物科技有限公司
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