PX459

PX330 and related PX459 plasmids are widely used for clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome editing. Screening for plasmids containing the correct sgRNA template insertion is one of the most important steps in this system. Different methods have been employed to screen sgRNA inserts. One such method is restriction enzyme (RE) mapping. In this study, the sgRNA template was initially cloned into the PX459 plasmid described above. Subsequently, the accuracy of the construct was determined by RE mapping. This method was established to screen PX459 plasmids harboring sgRNA. However, after ligating the sgRNA template into the RE-digested PX459 plasmid, a number of anomalies were detected. These data indicate that RE profiles are suitable only as an initial screen and that the identity of all plasmids with correctly identified RE profiles should be confirmed by Sanger sequencing.

Two sgRNA sequences were designed based on the available PAM sequences upstream of the γ-globin start codon. The sgRNA DNA template was then digested and cloned into the PX459 plasmid. To screen for correct sgRNA template insertion by RE mapping, the plasmid was cut with BbsI and EcoRI restriction enzymes. Digestion of plasmid PX459 with BbsI and EcoRI should produce three visible bands using gel electrophoresis. However, in recombinant PX459 containing the sgRNA template, the BbsI cleavage site was removed and only two bands were observed after BbsI-EcoRI double digestion. Several obtained recombinant PX459 plasmids with predicted two-band RE patterns were analyzed by Sanger sequencing. Most of these plasmids have correctly inserted sgRNA templates. However, many recombinant plasmids have been incorrectly validated based solely on their RE profiles.

Figure 1. A) Schematic representation of expected PX459-sgRNA restriction enzyme map. B) The BbsI cleavage site is removed in PX459 containing the sgRNA template insert. C) Digestion simulation of PX459 (PX459 with and without sgRNA) is shown. Using gel electrophoresis, double digestion of PX459 with EcoRI and BbsI should yield 3 visible bands (5253 bp, 3231 bp, 669 bp). In the PX459 plasmid with the sgRNA insert, the BbsI site was removed and only two bands (8509 bp, 669 bp) were observed. (Hassani M, et al. 2021)