Screening and characterization of integration sites based on CRISPR-Cpf1 in .

, a methylotrophic yeast, can utilize methanol as a carbon source and energy source to synthesize high-value chemicals, and is an ideal host for biomanufacturing. Constructing the . cell factory is somewhat impeded due to the absence of genetic tools for manipulating multi-gene biosynthetic pathways. To broaden its application in the field of metabolic engineering, this study identified and screened 15 novel integration sites in using CRISPR-Cpf1 genome editing technology, with EGFP serving the reporter protein. These integration sites have integration efficiencies of 10-100 % and varying expression strengths, which allow for selection based on the expression levels of genes as needed. Additionally, these integrated sites are applied in the heterologous biosynthesis of , such as the astaxanthin biosynthetic pathway and the carbon dioxide fixation pathway of the Calvin-Benson-Bassham (CBB) cycle. During the three-site integration process, the 8 genes of the CBB cycle were integrated into the genome of . This indicates the potential of these integration sites for integrating large fragments and suggests their successful application in metabolic engineering of . This may lead to improved efficiency of genetic engineering in .