Journal: Cells

Article Title: Efficiency and Fidelity of Site-Directed Mutagenesis with Complementary Primer Pairs

doi: 10.3390/cells15020138

Figure Lengend Snippet: QC2 mutagenesis with SuperFi II polymerase to engineer Cas9 mutants. ( A ) Domain organization of Cas9 (1.1) [ , ]. Five mutants to be engineered are indicated. Upstream from the coding sequence is the CAG promoter, a synthetic promoter composed of a CMV promoter, a β-actin promoter and an intron . The CAG promoter possesses GC-rich sequences that render the plasmid incompatible with the PCR conditions described for P3a mutagenesis, so the P3b PCR conditions were used instead . ( B ) Efficiency of QC2 mutagenesis via SuperFi II DNA polymerase. An expression plasmid for human Cas9 (1.1) [ , ], as depicted in panel A, was used as the PCR template under the P3b conditions . ( C ) Analysis of 6 plasmids sequenced for engineering the K562D mutant. Clone #6 possessed two insertions, which shared two repeats of a 27-nucleotide sequence (AGTGACCGTGGATCAGCTGAAAGAGGA), underlined in blue, identical to the primer site underlined in blue for clone #5. ( D ) Summary on the efficiency of QC2 mutagenesis in engineering 46 mutations on seven expression plasmids. The average efficiency reached 59.3%. The vector size ranged from 7.0 to 12.6 kb, with the GC-content reaching 95% for certain regions of the KAT2B and Cas9 expression vectors . Overall, QC2 mutagenesis outperforms the classical QuickChange mutagenesis method ( B) and P3 site-directed mutagenesis [ , ]. Although the efficiency is still lower than that from the P3a and P3b methods [ , ], QC2 mutagenesis is still reliable. The asterisk refers to the success rate of 46 mutants that we attempted to engineer for seven proteins: All but two (D1690A of p300, B; and Y189A of KAT2B, B) were successfully engineered. ( E ) Comparison of the four different mutagenesis methods. The P3a and P3b methods are much more efficient and faster than P3 mutagenesis [ , ]. The risk of introducing unwanted mutation is also much lower with the P3a and P3b methods. The P3 and QC2 methods frequently introduce unwanted insertions and deletions at the primer sites. Compared to the P3 method, QC2 is faster and more efficient, but it is still less efficient than the P3a and P3b methods. Second-site mutations refer to misincorporation during strand extension, which is mainly determined by the fidelity of the DNA polymerase used and distinct from primer-site insertion. The colony number refers to colonies obtained with 1 μL of a mutagenesis reaction mixture transformed into 10 μL of DH5α competent cells.

Article Snippet: For heat denaturation, 20 μL of the KAT2B or Cas9 expression plasmid (0.1 μg/μL with 1 × NEB restriction digestion buffer #2, Cat. B7002S, New England Biolabs, Ipswich, MA, USA) was incubated at 105 °C for 5 min before rapidly cooling on ice.

Techniques: Mutagenesis, Sequencing, Plasmid Preparation, Expressing, Comparison, Introduce, Transformation Assay