METHOD FOR PREPARING RANDOM SGRNA LIBRARY OF TARGET SEQUENCE BY MEANS OF ENZYMATIC METHOD
Provided in the present invention is a method for preparing a random sgRNA library of a target sequence by means of an enzymatic method. The preparation method comprises the steps of: nicking a PAM region of the target sequence of sample DNA with a random nickase Nt.CviPII; linking the fragmented DN...
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| creator | LIU, Qian SUN, Rui WANG, Man SONG, Dongliang HUANG, Cheng CHEN, Jingjing CAO, Zhen HOU, Ce |
| description | Provided in the present invention is a method for preparing a random sgRNA library of a target sequence by means of an enzymatic method. The preparation method comprises the steps of: nicking a PAM region of the target sequence of sample DNA with a random nickase Nt.CviPII; linking the fragmented DNA to a linker magnetic bead containing a BbsI enzyme cleavage site; performing cleavage with BbsI to release DNA; performing random primer extension; linking same to an sgRNA skeleton containing an MmeI enzyme cleavage site; performing cleavage with MmeI to release a protospacer region; linking same to a T7 promoter sequence; performing amplification to obtain an sgRNA library template containing the T7 promoter; and finally obtaining the sgRNA library by means of in vitro transcription. According to the method for preparing a random sgRNA library of a target sequence by means of an enzymatic method in the present application, all sgRNA groups randomly covering a target region can be prepared at one time, and the m |
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The preparation method comprises the steps of: nicking a PAM region of the target sequence of sample DNA with a random nickase Nt.CviPII; linking the fragmented DNA to a linker magnetic bead containing a BbsI enzyme cleavage site; performing cleavage with BbsI to release DNA; performing random primer extension; linking same to an sgRNA skeleton containing an MmeI enzyme cleavage site; performing cleavage with MmeI to release a protospacer region; linking same to a T7 promoter sequence; performing amplification to obtain an sgRNA library template containing the T7 promoter; and finally obtaining the sgRNA library by means of in vitro transcription. 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The preparation method comprises the steps of: nicking a PAM region of the target sequence of sample DNA with a random nickase Nt.CviPII; linking the fragmented DNA to a linker magnetic bead containing a BbsI enzyme cleavage site; performing cleavage with BbsI to release DNA; performing random primer extension; linking same to an sgRNA skeleton containing an MmeI enzyme cleavage site; performing cleavage with MmeI to release a protospacer region; linking same to a T7 promoter sequence; performing amplification to obtain an sgRNA library template containing the T7 promoter; and finally obtaining the sgRNA library by means of in vitro transcription. 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| subjects | BEER BIOCHEMISTRY CHEMISTRY COMBINATORIAL CHEMISTRY COMPOSITIONS THEREOF CULTURE MEDIA ENZYMOLOGY LIBRARIES, e.g. CHEMICAL LIBRARIES, IN SILICOLIBRARIES METALLURGY MICROBIOLOGY MICROORGANISMS OR ENZYMES MUTATION OR GENETIC ENGINEERING PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS SPIRITS VINEGAR WINE |
| title | METHOD FOR PREPARING RANDOM SGRNA LIBRARY OF TARGET SEQUENCE BY MEANS OF ENZYMATIC METHOD |
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