Prykhozhij et al., 2018 - Zebrafish knock-ins swim into the mainstream. Disease models & mechanisms   11(10) Full text @ Dis. Model. Mech.

Fig. 1.

Comparison of different study results using oligos for point mutation knock-ins. The figure panels show the Cas9-sgRNA CRISPR complex cutting genomic DNA and subsequent homology-directed repair by resection and knock-in mutation insertion. Synthesis-dependent strand-annealing (SDSA) is the DNA repair process involved in generating knock-ins when an ssODN (oligo) is present. (A) The basic strategy of point mutation knock-ins. The first step includes the identification of a functional sgRNA to couple with the Cas9 nuclease and direct it to the genomic site of choice. Second, the donor oligo with the mutation of interest and mutation(s) in sgRNA site or PAM is designed. Mutating the sgRNA homology site and/or the PAM site prevents subsequent rounds of Cas9-induced cuts of the edited genomic site. Third, upon the Cas9-induced break in genomic DNA, homology-dependent repair using the provided oligo can occur and the mutation is inserted into the genome. (B) The results of studies employing a comparison of ‘NT 126 S’ (sense symmetric) and ‘T 126 A right’ (anti-sense asymmetric) oligo knock-in efficiencies in zebrafish and in vitro (Prykhozhij et al., 2018b; Richardson et al., 2016). (C) The results of a cell culture study demonstrating which types of asymmetric oligos are more efficient (Liang et al., 2016). (D) The results of the study in this issue (Boel et al., 2018) that shows that symmetric oligos such as ‘NT 120 S’ perform nearly as well as two of the asymmetric oligos (‘NT 120 A left’ and ‘T 120 A right’), which, in turn, perform much better than their counterparts ‘NT 120 A right’ and ‘T 120 A left’.

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