CRISPR-Cas9 Knock In

CRISPR-Cas9 is a simple and efficient genome editing tool. Gene knock in cell lines can be generated conveniently by the CRISPR-Cas9 system. Our company offers genome-wide CRISPR gene knock in service. The selection markers in the donor cassette are FP genes or resistance genes. Two types of CRISPR knock in are offered, differing in repair mechanism that is used to achieve gene knock in, homology based or non-homology based. HDR-mediated knock in is achieved via homology directed repair. Another gene knock in is achieved via non-homology based repair (NHEJ).

The CRISPR-Cas9 complex has appeared as the most straightforward and simple way to induce targeted DNA DSBs and thus to activate cell repair mechanisms and stimulate knock in (KI). DSB repair can be error-prone generating small insertions and deletions (indels) but can also favour the insertion of exogenous DNA molecules either randomly or using homology arms. Programmable nucleases have thus been recognized as promising tools to knock in genetic elements and to allow in frame insertions, the goal of which can be to fuse a tag to the open reading frame (ORF) of interest or to precisely introduce a desired mutation, for example identified in patients.

CRISPR-Cas9 Knock In Contains The Following Sections

Mechanism of CRISPR-Cas9 Knock In

The advent of CRISPR-Cas9 technology has changed the landscape of gene targeting. The customizable nuclease is efficient in generating double-strand breaks (DSB) in the genome that can be used to knock in a DNA sequence at a specific locus in the genome in a number of species. Non-homologous end joining (NHEJ), which usually leads to a change in the nucleotide sequence, and homology-directed repair (HDR) are the two main mechanisms responsible for DNA repair after nucleases generate DSB at the target site, where NHEJ would lead to knockout (KO) characterized by unpredictable insertions or deletions (indels), whereas HDR results in knock in events, when a donor vector is co-introduced.

Mechanism of CRISPR-Cas9 Knock In

Fig 1. Mechanism of CRISPR-Cas9 Knock In.

NHEJ is a highly active DNA repair mechanism within the cell. Higher efficiency and insertion rate of NHEJ-based knock in can be used to circumvent the limitations of HDR-dependent mechanisms, while reconstituting the integrity of the targeted locus. NHEJ can ligate the broken DNA double strands without requiring a template, and therefore frequently introduces small insertions and deletions (indels). For the generation of loss-of-function alleles, indels within the open reading frame of a gene that cause frameshifts and subsequently lead to premature stop codons are usually selected. In parallel, various other alleles like single amino acid substitutions or deletions or bigger insertions or deletions potentially representing hypomorphic alleles can be isolated.

Studies have shown that inhibiting NHEJ or enhancing HDR via small molecules will improve the nuclease-mediated homologous recombination (HR) efficiency. The cleanest and most obvious technique for in frame knock in (KI) is to trigger by homologous recombination (HR) the insertion of a donor DNA flanked by homologous sequences to both sides of the break in the locus of interest. The CRISPR-Cas9 complex has appeared as the most straightforward and simple way to induce targeted DNA DSBs and thus to activate cell repair mechanisms and stimulate knock in (KI).

CRISPR-Cas9 Knock In Related References

1. T.O. Auer, F. Del Bene. CRISPR/Cas9 and TALEN-mediated knock-in approaches in zebrafish. Methods. 2014 Sep;69(2):142-50.
2. Albadri et al. Genome editing using CRISPR/Cas9-based knock-in approaches in zebrafish. Methods. Mar 12, 2017;1-24.
3. Auer et al. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. CSHL Press. 2013;24:142–153.