{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,17]],"date-time":"2026-01-17T03:23:31Z","timestamp":1768620211923,"version":"3.49.0"},"reference-count":58,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T00:00:00Z","timestamp":1766016000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000024","name":"Canadian Institutes of Health Research","doi-asserted-by":"crossref","award":["PJT-190308"],"award-info":[{"award-number":["PJT-190308"]}],"id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","doi-asserted-by":"publisher","award":["RGPIN-2019-07122"],"award-info":[{"award-number":["RGPIN-2019-07122"]}],"id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100013020","name":"Compute Canada","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100013020","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>GC-rich sequences affect DNA replication, recombination and repair, as well as RNA transcription in vivo. Such sequences may also impede site-directed mutagenesis in vitro. P3a site-directed mutagenesis is a highly efficient method, but it has not been tested with plasmids possessing GC-rich sequences. Here we report that it is very efficient with a BRPF3 expression vector but unsuccessful with that for KAT2B. Because two GC-rich regions located within the synthetic CAG promoter and the KAT2B coding region may form guanine (G)-quadruplexes and hinder plasmid denaturation during PCR, we developed P3b site-specific mutagenesis, achieving an average efficiency of 97.5% in engineering ten KAT2B mutants. Importantly, deletion mutagenesis revealed that either of the two GC-rich regions is sufficient for rendering the plasmid incompatible with P3a mutagenesis. Consistent with this, only P3b mutagenesis worked efficiently with several widely used sgRNA\/Cas9 expression vectors, which contain the CAG promoter, and with an expression vector for CDK13, which possesses an intrinsically disordered domain encoded by a GC-rich DNA fragment. Thus, this study highlights serious challenges posed by GC-rich sequences to site-directed mutagenesis and provides an effective remedy to address such challenges. The findings support that G-quadruplex formation is one mechanism whereby such sequences impede regular PCR-based mutagenesis methods.<\/jats:p>","DOI":"10.3390\/cells14242016","type":"journal-article","created":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T10:06:56Z","timestamp":1766052416000},"page":"2016","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Highly Efficient Site-Specific and Cassette Mutagenesis of Plasmids Harboring GC-Rich Sequences"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-7125-244X","authenticated-orcid":false,"given":"Paulina","family":"Varela-Castillo","sequence":"first","affiliation":[{"name":"Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A 1A3, Canada"},{"name":"Department of Medicine, McGill University, Montreal, QC H3A 1A3, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ethan","family":"Zhou","sequence":"additional","affiliation":[{"name":"Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A 1A3, Canada"},{"name":"Department of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7576-8087","authenticated-orcid":false,"given":"Arezousadat","family":"Razavi","sequence":"additional","affiliation":[{"name":"Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A 1A3, Canada"},{"name":"Department of Medicine, McGill University, Montreal, QC H3A 1A3, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elham","family":"Ebrahimi","sequence":"additional","affiliation":[{"name":"Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A 1A3, Canada"},{"name":"Department of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0598-120X","authenticated-orcid":false,"given":"Xiang-Jiao","family":"Yang","sequence":"additional","affiliation":[{"name":"Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A 1A3, Canada"},{"name":"Department of Medicine, McGill University, Montreal, QC H3A 1A3, Canada"},{"name":"Department of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada"},{"name":"Department of Medicine, McGill University Health Center, Montreal, QC H3A 1A3, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"6551","DOI":"10.1016\/S0021-9258(19)46967-6","article-title":"Mutagenesis at a specific position in a DNA sequence","volume":"253","author":"Hutchison","year":"1978","journal-title":"J. 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