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CRISPR/Cas Genome Editing: Reagent Selection and Experimental Design

In 2013, two papers published in Science (1,2) and one in eLIFE (3) demonstrated how the bacterial CRISPR/Cas system can be adapted to serve as a versatile platform for RNA-directed genome editing in mammalian cells. Since that time, the CRISPR system has been established as a groundbreaking technology with the potential to supplant the incumbent zinc finger and TALE nuclease approaches (i.e. ZFNs and TALENs, respectively). Given the central role that transfection plays in the genome editing process, we provide a protocol below illustrating how Mirus TransIT® Transfection Reagents could be used to deliver CRISPR/Cas plasmids.


Genome Editing: Reverse genetics experiments increasingly rely on DNA endonucleases that have been programmed to recognize and cleave specific locations in the genome. Following cleavage, two endogenous repair mechanisms, non-homologous end joining (NHEJ) and homology-directed repair (HDR) are triggered in response to the DNA break. The features of these DNA break repair pathways can be exploited to generate gene knock-outs or introduce defined modifications at the site of cleavage. NHEJ is an error-prone process that frequently results in the formation of small insertions and deletions that disrupt gene function. HDR requires homologous DNA as a template for repair and can be leveraged to create a limitless variety of modifications specified by the introduction of donor DNA containing the desired sequence flanked on either side by sequences bearing homology to the target.

CRISPR/Cas Systems: Bacteria and archaea exhibit chromosomal elements called clustered regularly interspaced short palindromic repeats (CRISPR) that are part of an adaptive immune system that protects against invading viral and plasmid DNA. In Type II CRISPR systems, CRISPR RNAs (crRNAs) function with trans-activating crRNA (tracrRNA) and CRISPR-associated (Cas) proteins to introduce double-stranded breaks in target DNA (4). Target cleavage by Cas9 requires base-pairing between the crRNA and tracrRNA as well as base pairing between the crRNA and the target DNA. Target recognition is facilitated by the presence of a short motif called a protospacer-adjacent motif (PAM) that conforms to the sequence NGG.

CRISPR Systems for Genome Editing: Several studies have shown that this system can be harnessed for genome editing in mammalian cells (1-3). Cas9 is normally programmed by a dual RNA consisting of the crRNA and tracrRNA. However, the core components of these RNAs can be combined into a single hybrid ‘guide RNA’ for Cas9 targeting (Figure 1). The use of a noncoding RNA guide to target DNA for site-specific cleavage promises to be significantly more straightforward than existing technologies such as ZFNs and TALENs. Using the CRISPR/Cas strategy, retargeting the nuclease complex only requires introduction of a new RNA sequence and there is no need to reengineer the specificity of protein transcription factors.
RNA-programmed DNA Cleavage by Cas9

Figure 1. Cas9 is targeted to DNA by a guide RNA that forms base pairs to itself and to the DNA target. Cleavage occurs on both strands (red X) 3 bp upstream of the PAM (1-4).


Genome editing experiments follow three basic steps:
  1. Design plasmids to target the desired genomic location.
  2. Deliver the plasmids by co-transfection.
  3. Analyze the transfected cells for the intended genomic modifications.
This protocol provides a framework for CRISPR/Cas plasmid design and delivery with Mirus TransIT® Transfection Reagents.


Cas9 Plasmid: Clone the Streptococcus pyogenes Cas9 gene into the appropriate expression vector. The Cas9 sequence should be codon optimized for expression in mammalian cells and fused to an epitope tag for assessing transfection efficiency and expression as well as one or more nuclear localization signals (NLSs) to target Cas9 to the nucleus (Table 1, Figure 2). Cas9 can be used in one of two ways. If both nuclease domains are intact, Cas9 will generate double-stranded breaks in the target. It is also possible to inactivate one of the catalytic domains of Cas9 in order to favor HDR over NHEJ which may reduce cellular toxicity. The D10A mutation converts Cas9 to a nickase that generates single stranded breaks at the desired site and has been used to lower the rate of NHEJ without effecting HDR (2).

Target Plasmid: Select a target sequence that is 20 nucleotides long and flanked on the 3' end by the sequence NGG. Use a BLAST search to ensure that the sequence is unique to avoid off-target cleavage events. Insert the 20 nucleotide target sequence and the CRISPR guide sequence into a vector under the control of the human U6 RNA polymerase III promoter. The target sequence should be attached to the 5' end of the CRISPR guide. A 5’ G is required for transcription initiation from the U6 promoter. Note that it is also possible to express full length crRNA and tracrRNA separately (1).

Donor Plasmid: For gene insertion, gene replacement and other sequence integrations, construct a plasmid containing left and right homology arms surrounding the sequence to be inserted at the cleavage site. As a starting point, choose homology arms roughly 0.5 to 1 kb in length. Several homology arm sizes may need to be tested to achieve efficient transfection and recombination.

The success of genome editing experiments depends on the efficiency of each step. Deliver the CRISPR/Cas plasmids under optimized cell culture and transfection conditions to ensure the best outcome.

Reagent Selection: Each cell type responds differently to a given transfection method. Choose the appropriate transfection reagent for your cell type (Table 2). For information on what reagent to choose, visit our transfection database, Reagent Agent® - a tool designed to help you determine the best delivery solution for any cell type including hard-to-transfect cell lines and primary cells.

Transfection Conditions: Cell confluency, reagent volume, and post-transfection incubation time are a few key parameters that impact the outcome of transfection experiments. For more information on getting the most out of your transfections, visit our Tips from the Bench section on Optimizing DNA Transfections.

Plasmid DNA Transfection Protocol: The following procedure describes how to perform plasmid DNA transfections using TransIT-X2® Dynamic Delivery System in 6-well plates. If using vessels with different surface areas, scale accordingly. For more details on performing transfections with TransIT-X2®, please view the full protocol.

A. Plate cells
  1. Approximately 18-24 hours before transfection, plate cells in 2.5 ml complete growth medium per well in a 6-well plate.
    Ideally cells should be ≥80% confluent prior to transfection.
    For adherent cells: Plate cells at a density of 0.8 - 3.0 × 105 cells/ml.
    For suspension cells: Plate cells at a density of 2.5 - 5.0 × 105 cells/ml.
  2. Incubate cell cultures overnight.
B. Prepare TransIT-X2®:DNA complexes (Immediately before transfection)
  1. Warm TransIT-X2® to room temperature and vortex gently before using.
  2. Place 250 µl of Opti-MEM® I Reduced-Serum Medium in a sterile tube.
  3. Add 2.5 µg (total) of combined plasmid DNA.
  4. Pipet gently to mix completely.
  5. Add 7.5 µl TransIT-X2® to the diluted DNA mixture.
  6. Pipet gently to mix completely.
  7. Incubate at room temperature for 15-30 minutes to allow sufficient time for complexes to form.
C. Distribute the complexes to cells in complete growth medium
  1. Add the TransIT-X2®:DNA complexes (prepared in Step B) drop-wise to different areas of the wells.
  2. Gently rock the culture vessel back-and-forth and from side-to-side to distribute the TransIT-X2® Reagent:DNA complexes.
  3. Incubate for 24-72 hours. It is not necessary to replace the complete growth medium with fresh medium.
Dilution Cloning and Screening: Cas9 activity can be detected using SURVEYOR® Mutation Detection Kits (Transgenomic®) followed by gel electrophoresis to detect cleavage fragments. Surveyor assays utilize the Cel-1 nuclease that recognizes and cleaves DNA mismatches that result from hybridization of wild-type and mutant sequences. Clonal populations can be isolated by limiting dilution, and the nature of the resulting mutations can be determined by standard or next generation sequencing methods.
Table 1. Key Sequences for CRISPR/Cas Plasmid Construction
Element Relevant DNA or Amino Acid Sequence

For the most updated Cas9 sequence, visit
Plasmids Used in CRISPR Genome Editing
Figure 2. Plasmids Used in CRISPR Genome Editing
Table 2. Mirus Plasmid DNA Delivery Solutions
Reagent Description Key Features
TransIT-X2® Broad Spectrum A novel, polymeric system for efficient delivery of plasmid DNA and/or siRNA/miRNA
TransIT®-2020 Broad Spectrum A low toxicity transfection reagent that exhibits superior performance in a wide range of cells including hard-to-transfect and primary cells
TransIT®-LT1 Broad Spectrum Our most popular high-efficiency, low-toxicity transfection reagent
TransIT® Cell Type Specific Reagents Cell Type Optimized Specifically developed for optimal delivery in certain cell types
Ingenio® Electroporation Solution Universal Electroporation Solution Deliver DNA to almost any cell type with a single solution. Compatible with most electroporation devices.

  1. Cong et al. (2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. Science
  2. Mali et al. (2013). RNA-guided human genome engineering via Cas9. Science
  3. Jinek et al. (2013). RNA-programmed genome editing in human cells. eLife
  4. Jinek et al. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science

Are you performing CRISPR experiments? Mirus is currently providing free samples of our TransIT® Transfection Reagents to labs interested in using them for CRISPR applications. Request a sample >>