PCR‐Free Site‐Directed Mutagenesis on Repetitive Sequences Using Single‐Stranded DNA‐Assisted Double‐Stranded DNA Nicking by DNAzymes

Abstract Site‐directed mutagenesis (SDM) is a key driver for many biochemical investigations and biotechnological applications. Despite decades of development, it is still difficult to perform SDM on some sequences, including highly repetitive ones, because polymerase chain reaction (PCR) is often used as the most effective method for SDM, but results in complicated products with these sequences. To overcome this limitation, we report herein a PCR‐free SDM method that uses DNA‐cleaving DNAzymes. Since these DNAzymes lack the capability of cleaving double‐stranded DNA, we employed single‐stranded DNAs as assisting DNAs to open superhelical plasmids, allowing the DNA‐cleaving DNAzymes to cleave the plasmid. Such a system, named single‐stranded DNA‐assisted double‐stranded DNA nicking by DNAzymes (DANDA), expanded the substrate scope of DNAzymes to double‐stranded, superhelical plasmids. We showed that DANDA is highly customizable and target‐specific, which allows successful generation of mutations on a plasmid containing PCR‐incompatible repetitive sequences. By using solely unmodified DNA oligos that are more cost‐effective and easier to prepare than other systems that employ either PNA or restriction enzymes, this DANDA system further expands the applications of DNA‐cleaving DNAzymes in synthetic biology for different biochemical and biotechnological applications.