Figure 1a: CRISPR/Cpf1-mediated mutant mouse breeding using CRISPR/Cpf1 to disrupt the function of the Foxn1 gene or the Tyrosinase gene. These mutations resulted in hairless mice and white hairy mice, respectively. Figure 1b: Schematic representation of delivery of Cpf1 RNP to mouse embryos by electroporation.
As a new tool for editing the CRISPR genome, Cpf1 has attracted wide attention because of its different nature from Cas9. It requires only a single RNA, cryRNA (CRISPR RNA), and thus assembly is simpler; its interleaved cleavage pattern may facilitate the replacement of existing DNA sequences with the desired sequence; it recognizes thymine-rich DNA sequences, and The sequence of guanine-rich sequences recognized by Cas9 is rarely discussed. In conclusion, Cpf1 is expected to expand the scope of the CRISPR genome editing target site with better editing efficiency.
In a new study, a team from the IBS Genome Editor successfully introduced mutations mediated by Cpf1 ribonucleoprotein (RNP, editorial: assembled by crRNA and Cpf1) into mouse embryos. Mutant mice. The target genes they chose to mutate are Foxn1 (a transcription factor that regulates the immune system that promotes skin hair growth) and Tyrosinase (which encodes tyrosinase, which catalyzes melanin production, in which melanin is a natural pigment that determines the skin's colour). The results of the study were published online June 6, 2016 in the journal Nature Biotechnology, entitled "Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins".
HUR K Junho, the first author of the paper, said, "These data suggest that transporting Cpf1 RNPs into mouse embryos results in mutations in target genes that disrupt their function, with mutation rates of 64% and 33%, respectively. We will mutate small Mouse embryos were transplanted into surrogate mothers to obtain mice carrying specific mutations. These mutations resulted in hairless mice and white hair mice, respectively (Fig. 1a).
Hur added, "To investigate whether Cpf1 has off-target effects, we performed genome-wide sequencing of genomic DNA isolated from a mouse with a mutation in the Foxn1 gene and its wild-type close relatives. Sequence analysis showed no off-target mutations. The occurrence of targeted deep sequencing of DNA from other mutant mice also showed no off-target mutations."
In this study, the researchers used electrical pulses to simultaneously infiltrate Cpf1 RNP into up to 50 mouse embryos (Fig. 1b). This new electroporation technology delivers Cpf1 RNP for genome editing to mouse embryos to produce mutant mice. This electroporation method is easy to carry out, fast, and scalable compared to conventional microinjection techniques.
After adding Cpf1 to the CRISPR editing toolbox, CRISPR editing techniques can be used to build a multitude of model mice. KIM Jin-Soo, author of the paper and author of the IBS Genome Editor, commented, "Given this study demonstrates the superior specificity of Cpf1, this new endonuclease will be more widely used for precise genome editing, while There are no unwanted mutations. The application of CRISPR/Cpf1 will be unlimited, from non-toxic anticancer drugs and stem cell treatments to high value-added livestock and agricultural products, and has always been open."
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