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The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/Cas) system is currently a widely used gene editing system. Its principle is that the gRNA produced by CRISPR transcription mediates Cas nuclease to target the target sequence and cut the sequence.
Figure 1 Schematic diagram of CRISPR/Cas9 gene editing (Source: Wellcome Trust SangerInstitute, Sanger)
In the CRISPR/Cas9 system, sgRNA (small guide RNA) recognizes and binds to the target sequence of the target gene, guiding Cas9 to cut the binding site to produce a DNA double-strand break (DSB). DSBs are repaired by non-homologous end joining (NHEJ). The proteins involved in the repair often insert or delete several bases at the end of the DNA. The repaired genes will lose their functions due to mutations, thus achieving the goal of repair in the body. Gene knockout.
Application: establishment of gene knockout cell lines, establishment of gene knockout animal disease models.
Technical advantages: Compared with RNAi, which "knocks down" the target gene at the mRNA level, the CRISPR/Cas9 system causes the deletion of the gene sequence, thereby completely silencing (i.e. knocking out) the target gene.
In the CRISPR/Cas9 system, sgRNA (small guide RNA) recognizes and binds to the target sequence of the target gene, guiding Cas9 to cut the binding site to generate a DNA double-strand break (DSB). Homologous recombination (HR) repair method introduces exogenous donor DNA into the target site of the genome to achieve gene knock-in.
Application: Establishment of gene fragment knock-in cell lines, gene single-base mutation cell lines, and gene knock-in to establish animal disease models.
Technical advantages: simple operation, high efficiency, broad spectrum, and provides BSL-1 and BSL-2 virus injection and experimental operation platforms.
The CRISPR-dCas9 system is a fusion of dCas9 with a transcriptional activator (such as VP64) or a transcriptional repressor (such as KRAB), and then combined with sgRNA can promote or inhibit the expression of the target gene.
Application: Overexpression of target genes in endogenous environment, induction of iPSCs, inhibition of expression, etc.
Technical advantages: simple operation, high efficiency, broad spectrum, and provides BSL-1 and BSL-2 virus injection and experimental operation platforms, and can be combined with RNAi.
If you want to manipulate gene expression using the CRISPR-Cas system, please click on the chat window at the bottom right, or send an email to bd@braincase.cn to confirm the details with us.
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