CRISPR oligonucleotides and gene clips
A nucleic acid molecule, target gene technology, applied in genetic engineering, biochemical equipment and methods, DNA/RNA fragments, etc.
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example 1
[0286] Example 1: One-step synthesis of gRNA templates and efficient cell engineering workflow
[0287] summary
[0288] The CRISPR-Cas9 system offers innovative genome engineering applications. For genome editing, expression of Cas9, mature crRNA and tracrRNA or a single guide RNA (gRNA) is required. Elements of mature crRNA and tracrRNA or gRNA are usually constructed into Cas9 expression plastids or constructed into standard plastids driven by U6 promoter for mammalian expression. In this example, a novel method for the rapid synthesis of gRNA templates is described, which is a combination of gene synthesis and DNA fragment assembly techniques with >96% assembly accuracy. In other words, more than 96% of the assembled nucleic acid molecules were the desired assembly products. The method allows rapid synthesis of guide RNA (gRNA) by in vitro transcription using short DNA oligonucleotides. In conjunction with Cas9 protein delivery, Cas9 / gRNA complexes can be transfected i...
example 2
[0326] Example 2: Rapid and Efficient Mammalian Cell Engineering by Cas9 Protein Transfection
[0327] summary
[0328] The CRISPR-Cas9 system provides an efficient genome editing platform that enables innovative applications of mammalian cell engineering. However, delivery of Cas9 and synthesis of guide RNA (gRNA) remain steps that can limit overall efficiency and general ease of use. Here we describe a method for the rapid synthesis of gRNA and the delivery of the Cas9 protein / gRNA ribonucleoprotein complex (Cas9 RNP) into a variety of mammalian cells by liposome-mediated transfection or electroporation. Using these methods, nuclease-mediated single-target indels have been reported to be as high as 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSCs). When this approach was used for multigene targeting in Jurkat cells, it was found that approximately 93% and 65% of the resulting isolated cell lines achieved two-locus and three-locus indels, respectively....
Embodiment approach
[0375] 1. A method for preparing nucleic acid molecules, said method comprising carrying out polymerase chain reaction (PCR) in a reaction mixture containing (i) double-stranded nucleic acid segments and (ii) at least one oligo a nucleotide capable of hybridizing to a nucleic acid at one end of the double-stranded nucleic acid segment,
[0376] wherein said nucleic acid molecule is produced by said PCR reaction, and
[0377] wherein said product nucleic acid molecule contains a promoter suitable for in vitro transcription at or near one end.
[0378] 2. The method according to item 1, wherein the nucleic acid molecule produced by the PCR reaction encodes an RNA molecule of 35 to 150 nucleotides in length.
[0379] 3. The method according to item 1, wherein the nucleic acid molecule produced by the PCR reaction has a length of 70 to 150 base pairs.
[0380] 4. The method according to item 1, wherein the nucleic acid molecule produced by the PCR reaction encodes an RNA molecul...
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