Preparation method of target sequence random sgRNA full-coverage group
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A full-coverage, target-sequence technology, applied in DNA/RNA fragments, recombinant DNA technology, combinatorial chemistry, etc., to achieve the effect of less bias, simple production, and uniform coverage
Pending Publication Date: 2022-04-05
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
[0054] Example 1: Design of sgRNA backbone and flow of RPTS
[0055] In this example, the backbone of the traditional sgRNA is modified, and the enzyme cutting site MmeI or EcoP15I is incorporated, but the structure of the sgRNA and the binding of Cas9 are not changed. See the modified sgRNA sequence and structure Figure 1-Figure 3 shown.
[0056] The process of testing RPTS, the process is as follows Figure 4 Shown:
[0057] (1) Restriction enzyme cleavage that recognizes the PAM sequence:
[0058] Table 2 restriction enzyme digestion system
[0060] React overnight at 37°C. After the reaction, 160 μL of Ampure DNA beads (Beckman) was added to recover the fragmented DNA. 45 μL water for elution.
[0061] Table 3 end cu...
Embodiment 2
[0092] Example 2: RPTS preparation of 18S rRNA or 28S rRNA.
specific Embodiment approach
[0093] In this example, we used RPTS to prepare a random sgRNA library covering 18S rRNA or 28S rRNA. The specific implementation is as follows:
[0094] (1) Acquisition of DNA fragments
[0095] Table 10 reverse transcription system
[0096] components Dosage 293 cellular RNA 1μg 10μM reverse transcription primer 18S-R or 28S-R 1μL 10mM dNTPs 1 Reaction at 75°C for 5 minutes 5×FS Buffer 4μL 0.1M DTT 1μL SuperScript IV (Thermo) 2μL total capacity 20 μL
[0097] 15 minutes at 42°C, 15 minutes at 50°C, 15 minutes at 55°C, 15 minutes at 50°C, 15 minutes at 55°C, and 15 minutes at 70°C.
[0098] Table 11 PCR amplification system
[0099] components Dosage The above reverse transcription product 1μL 10μM 18S-F / R or 28S-F / R 5μL Phusion High-Fidelity PCR Master Mix 25 μL Add water to 50μL
[0100] After denaturation at 98°C for 3 min, library cycles were amplified by...
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Abstract
The invention provides a preparation method of a target sequence random sgRNA full-coverage group. The preparation method comprises the following steps: cutting a PAM region of a target sequence by using restriction enzyme; connecting an upper sgRNA skeleton; obtaining a sequence of a targeting original spacer region through a side-cut activity restriction enzyme site on the sgRNA skeleton; connecting a T7 promoter; performing amplification to obtain an sgRNA library with a T7 promoter; and carrying out in-vitro transcription to obtain a target sequence sgRNA library. The invention also discloses a preparation method of the sgRNA library of the ribosomal RNA, a method for removing the ribosomal RNA in the RNA library, and a method for removing a human whole genome from a host genome. The method disclosed by the invention has the advantages of low cost, simplicity in manufacturing, uniform coverage, small preference, no limitation by the length of a target sequence, no need of designing sgRNA on a large scale and the like.
Description
technical field [0001] The patent of the invention relates to a method for preparing a target sequence random sgRNA full coverage group, 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 to the sgRNA...
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