Preparation method of random sgRNA library of enzymic method target sequence
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A target sequence and library technology, which is applied in the field of enzymatic target sequence random sgRNA library preparation to achieve the effects of small preference, uniform coverage and low cost
Pending Publication Date: 2022-04-08
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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In the design and synthesis of sgRNA, both cost and technology are great challenges
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Embodiment 1
[0038] Example 1: ERSP process
[0039] (1) Preparation of magnetic beads containing Bbs linker
[0040] BbsI-F and BbsI-R were dissolved in 100mM NaCl to 100μM, mixed with equimolar concentration, reacted at 95℃ for 5min, and decreased by 1℃ per minute. After the reaction, the annealed linker was diluted to 10 μM with water. Take 20 μL of the diluted adapter, add 5 μL of streptavidin magnetic beads C1 (Thermo), and rotate and mix at room temperature for 30 min. Wash the magnetic beads 3 times with 100mM NaCl.
[0041] (2) Recognition of restriction enzyme cutting of PAM sequence:
[0042] Table 2 restriction enzyme digestion system
[0043] components Dosage human genomic DNA 1μg 10×rCutSmart buffer 1μL Nt.CviPII (NEB) 0.5-2U Hydrate to 10μL
[0044]React at 37°C for 30 minutes, react at 98°C for 10 minutes, and place on ice immediately. The effects of different input amounts of Nt.CviPII on DNA fragmentation are as follows: ...
Embodiment 2
[0076] Example 2: Application of ERSP on removal of rRNA and host genome.
specific Embodiment approach
[0077] In this example, we first prepared a random sgRNA library covering 18SrRNA and 28S rRNA using human RNA as a template according to the ERSP method in Example 1; using human genomic DNA as a template, prepared a random sgRNA library covering human genomic DNA Library, referred to as ERSP library, and applied to rRNA removal and host genome removal. The specific implementation is as follows:
[0078] Table 10 sgRNA library and Cas9 protein pre-assembly:
[0082] components Dosage Cas9-sgRNA library 5μL RNA or DNA library 1-100ng 10×NEB buffer 3.1 1μL total capacity 10μL
[0083] 37°C for 30-90min, 90°C for 10min.
[0084] (5) Library amplification
[0085] Table 12
[0086] components Dosage The above re...
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Abstract
The invention provides a preparation method of an enzymic method target sequence random sgRNA library. The preparation method is characterized by comprising the following steps: nicking a PAM region of a sample DNA target sequence by adopting a random nicking enzyme Nt.CviPII; linker magnetic beads containing BbsI restriction enzyme cutting sites are connected with the fragmented DNA; carrying out BbsI cleavage to release DNA; performing random primer extension; connecting an sgRNA skeleton containing an MmeI restriction enzyme cutting site; performing MmeI cutting to release the original interval region; connecting a T7 promoter sequence; performing amplification to obtain an sgRNA library template containing the T7 promoter; and carrying out in-vitro transcription to obtain the sgRNA library. According to the preparation method of the enzyme-method target sequence random sgRNA library, all sgRNA groups which randomly cover a target area can be prepared at one time, and the preparation method has the advantages that the cost is low, the preparation is simple, the coverage is uniform, the preference is small, the method is not limited by the length of the target sequence, the sgRNA does not need to be designed on a large scale and the like, and has an important application value in capture and removal of target genes.
Description
technical field [0001] The patent of the invention relates to a method for preparing a random sgRNA library of target sequences by enzymatic method, which belongs to the field of biotechnology. Background technique [0002] Since its development, CRISPR gene editing technology has been widely used in various fields such as gene therapy, in vitro diagnosis, gene capture and target gene removal, and won the 2020 Nobel Prize in Physiology and Medicine. It is an efficient and practical technology. The practical CRISPR system is mainly composed of two parts, one is the Cas protein with two endonuclease active sites, which is responsible for cutting the two strands of DNA at the target site; the other is the DNA pairing sequence with the target site The guide RNA (sgRNA) that binds to the Cas protein sequence is responsible for recruiting the Cas protein and guiding the Cas protein to bind to the complementary paired target site. In the CRISPR system, the Cas protein first binds ...
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