title: A Vector with a Single Promoter for In Vitro Transcription and Mammalian Cell Expression of CRISPR gRNAs.
abstract: The genomes of more than 50 organisms have now been manipulated due to rapid advancement of gene editing technology. One way to perform gene editing in the mouse using the CRISPR/CAS system, guide RNA (gRNA) and CAS9 mRNA transcribed in vitro are microinjected into fertilized eggs that are then allowed to develop to term. As a rule, gRNAs are tested first in tissue culture cells and the one with the highest locus-specific cleavage activity is chosen for microinjection. For cell transfections, gRNAs are typically expressed using the human U6 promoter (hU6). However, gRNAs for microinjection into zygotes are obtained by in vitro transcription from a T7 bacteriophage promoter in a separate plasmid vector. Here, we describe the design and construction of a combined U6T7 hybrid promoter from which the same gRNA sequence can be expressed. An expression vector containing such a hybrid promoter can now be used to generate gRNA for testing in mammalian cells as well as for microinjection purposes. The gRNAs expressed and transcribed from this vector are found to be functional in cells as well as in mice.
authors: Mouse Genetics Core Facility, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.|Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.
authors: Peter J
authors: PJ
authors: Romanienko
authors: Mouse Genetics Core Facility, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.
authors: Joseph
authors: J
authors: Giacalone
authors: Mouse Genetics Core Facility, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.
authors: Joanne
authors: J
authors: Ingenito
authors: Mouse Genetics Core Facility, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.
authors: Yijie
authors: Y
authors: Wang
authors: Mouse Genetics Core Facility, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.
authors: Mayumi
authors: M
authors: Isaka
authors: Mouse Genetics Core Facility, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.
authors: Thomas
authors: T
authors: Johnson
authors: Mouse Genetics Core Facility, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.|Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.
authors: Yun
authors: Y
authors: You
authors: Mouse Genetics Core Facility, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.|Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.
authors: Willie H
authors: WH
authors: Mark
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases