title: Enhanced CRISPR/Cas9-mediated biallelic genome targeting with dual surrogate reporter-integrated donors.
abstract: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has recently emerged as a simple, yet powerful genome engineering tool, which has been widely used for genome modification in various organisms and cell types. However, screening biallelic genome-modified cells is often time-consuming and technically challenging. In this study, we incorporated two different surrogate reporter cassettes into paired donor plasmids, which were used as both the surrogate reporters and the knock-in donors. By applying our dual surrogate reporter-integrated donor system, we demonstrate high frequency of CRISPR/Cas9-mediated biallelic genome integration in both human HEK293T and porcine PK15 cells (34.09% and 18.18%, respectively). Our work provides a powerful genetic tool for assisting the selection and enrichment of cells with targeted biallelic genome modification.
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.|Department of Biology, Zun Yi Normal College, Zunyi, Guizhou, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Wu
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Xu
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Chonghua
authors.initials: C
authors.lastname: Ren
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Xinyi
authors.initials: X
authors.lastname: Li
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Huijiao
authors.initials: H
authors.lastname: Lv
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Furong
authors.initials: F
authors.lastname: Han
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Zehui
authors.initials: Z
authors.lastname: Wei
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Zhiying
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D001761:Bleomycin / Q000494:pharmacology; D064113:CRISPR-Cas Systems; D002460:Cell Line; D004351:Drug Resistance / Q000235:genetics; D005434:Flow Cytometry; D000072669:Gene Editing / Q000379:methods*; D016678:Genome / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D011691:Puromycin / Q000494:pharmacology; D015203:Reproducibility of Results; D013552:Swine; D000097573:Red Fluorescent Protein
chemical_list: D001761:Bleomycin; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011691:Puromycin; D000097573:Red Fluorescent Protein; C105427:Zeocin
keywords: CRISPR/Cas9; biallelic genome engineering; donor plasmid; homology directed repair; surrogate reporter