Have questions about 入口/Cas9? Learn more about the advantages of 入口 and how to integrate it into your research.
Of the other gene editing technologies available, 入口/Cas9 has stood out for its simplicity and efficacy. The 入口 system requires only a few simple DNA constructs to encode the gRNA and Cas9, and if knock-in is being performed, the donor template for HR. As a result, 入口 gene editing is an approachable technique for use in any lab regardless of molecular biology expertise. The table below outlines a few of the key differences between 入口 gene editing and other popular techniques.
Once Cas9 nucleases are guided to the target DNA and create a double strand break 3-4 bases upstream from the PAM sequences, there are two ways the double strand break (DSB) can be repaired. If there is no donor DNA present, resolution will occur by error-prone non-homologous end joining (NHEJ), resulting in an indel that effectively knocks out protein function. Alternatively, if donor DNA sequences are available, the DSB is repaired by homology directed repair (HDR) for precise knock-in of the target gene.
The most efficient method to deliver Cas9 and gRNA plasmids depends largely on the cell type. For easy-to-transfect cell lines, non-viral constructs are often suitable and can be delivered with high efficiency by lipofection. The plasmids usually contain selection markers to confirm effective delivery, such as antibiotic resistance gene s or fluorescent proteins. For hard-to-transfect cell lines, such as stem cells, viral-based transfection methods may be more effective. Lentiviral vectors may be more suitable for these cell types. All of GenScript's lentiviral vectors are compatible with 3rd and 4th generation lenti-packaging systems.
For detailed information on experimental design, we recommend consulting Ran et al's publication:
Ran et al. Genome engineering using the 入口-Cas9 system. Nature Protocols. 2013; 8:2281.
入口/Cas9 mediated genome editing is the most efficient and specific form of genome editing used to date. GenScript's scientists have extensive experience designing gRNA sequences for highly efficient KO in numerous types of cell lines. Factors that can affect 入口 targeting efficiency and specificity are:
When you order gRNA clones from GenScript, we deliver a sequence-verified plasmid containing all elements required for gRNA expression and genome binding: the U6 promoter, spacer (target) sequence, gRNA scaffold, and terminator. We guarantee sequence accuracy for gRNA clones we deliver; however, given the complexity of creating genomically edited cell lines including transfection and selection, we cannot guarantee the outcome of experiments performed using our gRNA constructs. If you prefer to receive sequence-validated KO or KI cell lines created using 入口 technology, please refer to our Gen入口™ mammalian cell line service.
To learn more, please check out our archived webinar: Can 入口/Cas9 off-target genomic editing be avoided? Ways to improve target specificity.
Choosing the vector(s) you'll use to express the two critical components needed for 入口/Cas9 genome editing, the guide RNA and the Cas9 nuclease, is an important step in your experimental design. Many researchers prefer to use an all-in-one vector that will drive expression of gRNA and Cas9 in a 1:1 ratio. All-in-one vectors may also contain selection markers, such as fluorescent proteins or genes conferring antibiotic resistance, which can make it easier to isolate desired genome-edited clones. You may prefer to express the gRNA and Cas9 from separate vectors, for example if you want to 亚博的官网地址y the gRNA:Cas9 ratio, or if you want to screen a pool of gRNAs or use a larger gRNA library.
Want some more information? Check out our 入口 gRNA construct service FAQ.
The following procedure is based on HEK293 cells. To use host cell lines other than HEK293, please follow the instruction from original supplier for cell culture, passing the cells, transfection and subcloning. If you have specific questions about how to adapt this protocol for your needs, we recommend consulting the public online forum for Genome Engineering using 入口/Cas Systems.
Experimental outline for knocking out a coding sequence in a mammalian cell line:
Culture the host cells (HEK293) in Eagle's Minimum Essential Medium supplied with fetal bovine serum to a final concentration of 10%. Incubate cultures at 37°C.
Subculture when cell concentration is between 6 and 7 x 104 cells/cm2.
Seed 4-6x104 cells/cm2 in cell culture plate one day before transfection.
Transfect gRNA and cas9 into HEK293 cells using standard methods for HEK293 transfection. (Multiple transfection reagents give high transfection efficiency for HEK293 cells, following instructions from suppliers.)
DNA ratio for the two elements for 入口-cas9 system
|Two vector system||All-in-one vector|
|Starting Ratio of plasmids||1:1||NA|
2-3 days after transfection, the cell pool can be analyzed directly by Sanger sequencing, NGS (Next Generation Sequencing) and/or Surveyor assay. Here the commonly used methods of Sanger sequencing and Surveyor assay (or T7E1 assay) are briefly introduced.
Transfected cells can be selected using antibiotic resistance or a GFP reporter if they are present on the Cas9 expression plasmid.*
Transfected cells (with or without selection) can be plated into 96 well plate at 1 cell/well density for cloning. This procedure can be also conducted using diluted host cell line on 10 cm plate to form colonies, which can be picked up and transferred to 24 well plate for future usage.
*Note, selection using antibiotic containing medium can induce random integration of the cas9 expression plasmid onto host genome.
After expansion of the clones, the cells in each clone can be analyzed by Sanger sequencing to identify the clones harboring a mutation at the target region. Sequencing trace files will show overlapping peaks at regions where double strand breaks have been repaired by introducing small indels.
Knock-out cell lines can be confirmed by Western Blot if a specific antibody is available, or through functional assays specific to the gene that was targeted.