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.
Highlights
Gene editing has become a valuable tool in the study of gene function across many species [1]
Without additional cloning one can use PCR and appropriate primers to synthesize an amplicon containing the same guide RNA (gRNA) sequence with T7 added as the promoter and perform in vitro transcription rather than make a new vector [9]. To avoid these multiple cloning steps, we have constructed a plasmid vector containing a single U6T7 hybrid promoter with an adjacent cloning site for insertion of a gRNA template. We show that this vector is capable of producing gRNAs by both the human U6 promoter (hU6) and the T7 promoters
Since cell transfection assays require gRNAs expressed from an hU6 promoter while gRNAs for microinjection are transcribed from a T7 promoter in vitro, two expression plasmids are needed to achieve these goals
Summary
Gene editing has become a valuable tool in the study of gene function across many species [1]. The most widely used approach is the CRISPR/CAS9 system derived from the adaptive immune response of bacteria [2]. To achieve genome modification by this technique, only two simple components are required: the CAS9 protein and a guide RNA (gRNA) containing 17–20 nucleotides of identity to a target sequence proximal to a protospacer adjacent motif (PAM). When gRNA forms a complex with the CAS9 nuclease, a DNA double strand break (DSB) will occur in the genomic target specified by the gRNA. For genomic manipulation of cells in culture, they are typically transfected with a single plasmid vector expressing the gRNA from an RNA Polymerase III promoter derived from the human snRNA U6 gene and the CAS9 mRNA from an RNA Polymerase II promoter [3].
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