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Although CRISPR-based gene editing systems have been established in <jats:italic>K. phaffii<\/jats:italic>, there are still some inadequacies compared to the model organism <jats:italic>Saccharomyces cerevisiae<\/jats:italic>. In this study, a redesigned gRNA plasmid carrying red and green fluorescent proteins facilitated plasmid construction and marker recycling, respectively, making marker recycling more convenient and reliable. Subsequently, based on the knockdown of <jats:italic>Ku70<\/jats:italic> and <jats:italic>DNA ligase IV<\/jats:italic>, we experimented with integrating multiple DNA fragments at a single locus. A 26.5-kb-long DNA fragment divided into 11 expression cassettes for lycopene synthesis could be successfully integrated into a single locus at one time with a success rate of 57%. A 27-kb-long DNA fragment could also be precisely knocked out with a 50% positive rate in <jats:italic>K. phaffii<\/jats:italic> by introducing two DSBs simultaneously. Finally, to explore the feasibility of rapidly balancing the expression intensity of multiple genes in a metabolic pathway, a yeast combinatorial library was successfully constructed in <jats:italic>K. phaffii<\/jats:italic> using lycopene as an indicator, and an optimal combination of the metabolic pathway was identified by screening, with a yield titer of up to 182.73\u00a0mg\/L in shake flask fermentation.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Key points<\/jats:title>\n            <jats:p>\n              <jats:italic>\u2022 Rapid marker recycling based on the visualization of a green fluorescent protein<\/jats:italic>\n            <\/jats:p>\n            <jats:p>\n              <jats:italic>\u2022 One-step multifragment integration and large fragment knockout in the genome<\/jats:italic>\n            <\/jats:p>\n            <jats:p>\n              <jats:italic>\u2022 A random assembly of multiple DNA elements to create yeast libraries in K. phaffii<\/jats:italic>\n            <\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Graphical Abstract<\/jats:title>\n          <\/jats:sec>","DOI":"10.1007\/s00253-024-13037-1","type":"journal-article","created":{"date-parts":[[2024,2,7]],"date-time":"2024-02-07T09:02:17Z","timestamp":1707296537000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["CRISPR\/Cas9-based toolkit for rapid marker recycling and combinatorial libraries in Komagataella phaffii"],"prefix":"10.1007","volume":"108","author":[{"given":"Wei","family":"Zhou","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuanyi","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guosong","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weichuang","family":"Qin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dongzhi","family":"Wei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fengqing","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1506-6198","authenticated-orcid":false,"given":"Bei","family":"Gao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,2,7]]},"reference":[{"issue":"12","key":"13037_CR1","doi-asserted-by":"publisher","first-page":"5301","DOI":"10.1007\/s00253-014-5732-5","volume":"98","author":"M Ahmad","year":"2014","unstructured":"Ahmad M, Hirz M, Pichler H, Schwab H (2014) Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. 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