bioarray123 authored 7 months ago

Knock-in mice are an important research model in which foreign genes or DNA fragments are introduced into the mouse genome and transmitted to the next generation. From the study of gene function to the production of therapeutic humanized antibodies, knock-in mice have a wide range of applications. Genome editing in mice initially relied on random integration of target vectors in embryonic stem cells, and then injection of target clones into donor mouse blastocysts. Later, engineered nucleases were developed to make genome editing more precise. Typical engineered nucleases include ZFN, TALEN and CRISPR. All three nucleases can induce double-strand breaks in the genome. Then, the repair pathway in the cell will be activated, which will eventually lead to knockout of the NHEJ gene or when the donor DNA is present, it is a knock-in mouse for HDR. As a state-of-the-art technology, CRISPR/Cas9 has become the main method for generating genome-edited mice.

The CRISPR/Cas9 genome editing method is the last new feature in the genetic toolbox. The most common version was developed by an adaptive immune defense bacterial system against the phage present in Streptococcus pyogenes. For this purpose, the complete synthesis of the nuclease reading frame is completed, thereby optimizing translation in mammalian cells. Importantly, unlike other nucleases used for genome editing, Cas9 uses short RNA as a guide to target genome sequences. This single guide RNA (sgRNA) is 100 nucleotides (nt) long, and its 5'end is chosen to be complementary to the target DNA strand to which it anneals.

If the 17-20nt DNA target sequence is followed by the so-called protospacer adjacent motif (PAM), Cas9 will cleave the target on both strands. The double-strand break located 3nt upstream of PAM will be repaired by NHEJ or HDR. NHEJ errors produce random indels, while HDR can be used to replicate the precise modifications present in the foreign DNA template. The key innovation is that the specificity of Cas9 for genomic targets is derived from the complementarity of sgRNA:DNA, rather than protein structure, without modification. Therefore, editing the selected genome sequence can avoid laborious protein design. Therefore, the method is simple, versatile, easy to reuse, and very efficient in most cases.

Genome editing relies on the ability of RNA-guided Cas9 nuclease to cut the two strands of DNA. Target 17-20nt genomic DNA (called protospacer). The DNA sequence is identified by Watson-Crick base pairing between the spacer component of a single guide RNA (ie its 5'end) and the complementary strand of the target DNA, annealing must be performed at PAM (5'-NGG- 3') upstream. NHEJ or HDR can repair double-strand breaks through cells. NHEJ is an error-prone process, usually resulting in a small number of insertions or deletions. HDR is another approach, requiring another complete copy of the sequence to be repaired in the nucleus. If an exogenous DNA template is provided, HDR allows the introduction of targeted modifications at the double-strand break site.

Another important application of CRISPR/Cas9 is to improve the efficiency of homologous recombination, which was previously only possible in mouse ES cells. When a large DNA construct is introduced into ES cells that contains sequences homologous to the genomic locus, it can spontaneously integrate into the target locus. The frequency of targeted integration increases with the size of the homologous sequence, but usually remains low. When the homology spans 10 kb, the typical frequency is 1% of DNA integration events. Using CRISPR/Cas9 to produce double-strand breaks in the target locus will cause this frequency to increase greatly. Therefore, by injecting Cas9 mRNA, sgRNA and circular plasmid constructs into mouse oocytes, the targeted insertion of large plasmid constructs can be achieved without using ES cells. However, it is reported that the HDR frequency of constructs with several thousand bases of homology exceeds 30%. HDR can also be obtained through very short homology and single-stranded oligonucleotides as templates. For the targeted integration of large constructs, an ingenious protocol suggests combining several CRISPR-mediated cleavage and single-stranded oligonucleotides, allowing for the optional introduction of any sequence between the construct and the targeted genomic locus Homology.

The CRISPR/Cas9 platform is committed to providing professional CRISPR/Cas9 gene-editing services, from gRNA design to customized CRISPR/Cas9 knock-in mouse services for F1 mice. Aiming at the Rosa26 knock strategy design, the CRISPR/Cas9 platform provides a comprehensive and effective point mutation strategy to maximize the efficiency of Cas9, including gRNA design and synthesis (targeting the Rosa26 locus sequence), donor vector construction, and PCR primers for genotype identification.

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