Abstract
Here, in this chapter, we describe a detailed protocol for the method named Cas9-mediated protein evolution reaction or short CasPER. CasPER is based on the generation of large 300-600-bp mutagenized linear DNA fragments by error-prone PCR which are used as a donor for repair of double-strand break mediated by Cas9 and subsequently integrated to the genome. This method can be efficiently used for directed evolution of desired essential or nonessential genes in the genome and most importantly can be multiplexed. Altogether, the described method allows for heterogeneous DNA integration with successful transformation efficiencies of 98-100% for both single and multiplex targeting.
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