Applications: Genome Editing Using CRISPR/Cas9

CRISPR/Cas9 Genome Editing:
pDNA + gRNA Transfection

TransIT-X2® Dynamic Delivery System for Plasmid DNA and Guide RNA Oligonucleotide Delivery

Cas9 protein and guide RNA can both be encoded as plasmid DNA for transfection. Alternatively, Cas9 can be delivered as plasmid DNA, and guide RNA can be supplied as an RNA oligonucleotide. Benefits to these approaches include:

Low Cost – Plasmid DNA is a renewable, cost-effective format
Flexibility – Cas9 and guide RNA plasmids are suitable for stable or transient transfection
Ease of use – Guide RNA oligonucleotide format enables simple retargeting of Cas9 to different loci

DNA/RNA Transfection Protocols

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CRISPR/Cas9 Delivery Methods – Cas9 and Guide RNA Plasmids. (A) Cas9 and guide RNA are encoded on the same plasmid. (B,C) Cas9 and guide RNA(s) are encoded on separate plasmids. (A,B) The wild-type Cas9 enzyme contains two endonuclease domains which cleave the target DNA on both strands when programmed with a guide RNA. (C) The D10A mutation converts Cas9 to a nickase that generates single-stranded breaks in the target DNA. For improved target specificity, Cas9 D10A can be used with paired guide RNAs targeting opposite strands to create staggered double-stranded breaks.

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CRISPR Plasmid and gRNA Delivery Approaches

CRISPR/Cas9 Delivery Methods – Cas9 Plasmid + Guide RNA Oligonucleotides. Cas9 is supplied as plasmid DNA, and guide RNA(s) are supplied as either synthetic or in vitro transcribed RNA oligonucleotides. (A) The wild-type Cas9 enzyme contains two endonuclease domains which cleave the target DNA on both strands when programmed with a guide RNA. (B) The D10A mutation converts Cas9 to a nickase that generates single-stranded breaks in the target DNA. For improved target specificity, Cas9 D10A can be used with paired guide RNAs targeting opposite strands to create staggered double-stranded breaks.

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High Efficiency CRISPR Genome Editing with TransIT-X2

Efficient Genome Editing with Cas9 Plasmid DNA + Guide RNA Oligonucleotides. HEK293T/17, U2OS and NHDF cells were co-transfected with 0.5 µg of Cas9 encoding pDNA (MilliporeSigma) and 50nM PPIB targeting 2-part gRNA (Dharmacon) using TransIT-X2® Dynamic Delivery System (2 µl/well of a 24-well plate, Mirus Bio). A T7E1 mismatch detection assay was used to measure cleavage efficiency at 48 hours post-transfection.

Glossary of CRISPR TermsClick to expand

Term	Definition
Cas9	CRISPR Associated Protein 9 - Cas9 is an RNA-guided DNA endonuclease from the type II CRISPR system of Streptococcus pyogenes that has been adapted for use in genome editing applications.
Cas9 Nickase	A Cas9 mutant that cuts one strand of double-stranded DNA Cas9 has two endonuclease domains which together cleave both strands of DNA. Mutations in either of these domains convert Cas9 to a "nickase" which cleaves only a single strand in the DNA target. Two Cas9 nickases combined with gRNAs that target opposite DNA strands can be used to create DSBs with fewer off-target effects.
CRISPR	Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR refers to prokaryotic DNA elements involved in adaptive immunity which are characterized by clusters of identical repeats interspaced with non-identical segments called spacers. CRISPR has evolved to refer more generally to the use of Cas9 for genome editing.
crRNA	CRISPR RNA - One of two RNAs required to form a functional gRNA. The crRNA contains the sequence complementary to the DNA target and a segment of RNA that base pairs with the tracrRNA.
DSB	Double Strand Breaks result from endonucleolytic cleavage of both strands of DNA. This can be achieved through the use of wild type Cas9 or by employing two Cas9 nickases targeting opposite DNA strands.
Genome Editing	The creation of desired genetic modifications including gene insertions, deletions, or replacements through the use of targeted nucleases such as Cas9.
gRNA	Guide RNAs bind to Cas9 and direct the complex to a specific genomic location. Naturally occurring guide RNAs consist of two parts: a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). Alternatively, the crRNA and tracrRNA can be combined into a single chimeric oligonucleotide called a single guide RNA (sgRNA).
HDR	Homology Directed Repair is a mechanism of DNA repair that uses a homologous DNA template to rebuild sites of genomic damage. HDR can be leveraged in genome editing experiments to make precise genomic alterations by supplying the desired sequence for insertion flanked by segments of DNA that are homologous to the sequence surrounding the Cas9-induced DSB.
InDel	(Insertion/Deletion) Following creation of a DSB by Cas9, the cell initiates DNA damage repair. Repair by the error-prone NHEJ pathway can result in small insertions and/ or deletions at the site of cleavage. These indels can cause frameshift mutations or premature stop codons resulting in a genetic knock-out.
Mismatch Assay	A method for detection of indel mutations following Cas9 cleavage. Targeted genomic DNA is amplified by PCR. The PCR products are melted and reannealed to allow heteroduplexes to form between wild-type and mutant DNA. The hybridized products are then incubated with an enzyme that cleaves heteroduplexes but not perfectly matched DNA. The resulting DNA fragments are analyzed by electrophoresis to determine the percentage of cleavage events that results from indel formation.
NHEJ	Non-Homologous End-Joining is the predominant DNA DSB repair mechanism in mammalian cells. Unlike HDR, NHEJ directly ligates the ends of the DSB and does not require a homologous repair template. Researchers capitalize on the error-prone nature of NHEJ to create indels following targeted cleavage with Cas9.
Off-target Effects	Off-target effects refers to Cas9 cleavage events at genomic locations other than the intended site.
PAM	Protospacer-Adjacent Motif - In the naturally occurring prokaryotic CRISPR/Cas system, the DNA sequences recognized by gRNA are called protospacers. The PAM is a short sequence next to the target site that is required for Cas9 targeting both in prokaryotic adaptive immunity and in mammalian genome editing experiments.
sgRNA	Single Guide RNA, a chimeric RNA composed of crRNA and tracrRNA, connected by a short RNA linker.
Target Sequence	A 20 nucleotide genomic DNA sequence which base-pairs with gRNA and is cleaved by Cas9.
tracrRNA	Trans-Activating crRNA - One of two RNAs required to form a functional gRNA. The tracrRNA forms base pairs with the crRNA and is required for Cas9-mediated target cleavage.

Plasmid DNA and Guide RNA Transfection Protocols

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CRISPR pDNA Transfection ProtocolClick to expand

CRISPR pDNA Transfection Workflow

Transfection Conditions: Cell confluency, reagent volume, and post-transfection incubation time are a few key parameters that affect 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

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.
Incubate cell cultures overnight.

B. Prepare TransIT-X2®:DNA complexes (Immediately before transfection)

Warm TransIT-X2® to room temperature and vortex gently before using.
Place 250 µl of Opti-MEM® I Reduced-Serum Medium in a sterile tube.
Add 2.5 µg (total) of combined plasmid DNA encoding Cas9 and/or guide RNA.
Pipet gently to mix completely.
Add 5 µl TransIT-X2® to the diluted DNA mixture.
Pipet gently to mix completely.
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

Add the TransIT-X2®:DNA complexes (prepared in Step B) drop-wise to different areas of the wells.
Gently rock the culture vessel back-and-forth and from side-to-side to distribute the TransIT-X2® Reagent:DNA complexes.
Incubate for 24-72 hours. It is not necessary to replace the complete growth medium with fresh medium.

CRISPR pDNA + gRNA Transfection ProtocolClick to expand

CRISPR Plasmid DNA Transfection Workflow

Plasmid DNA and RNA Oligonucleotide Transfection Protocol: The following procedure describes how to perform plasmid DNA and RNA oligonucleotide co-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

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.
Incubate cell cultures overnight.

B. Prepare TransIT-X2®:DNA:RNA complexes (Immediately before transfection)

Warm TransIT-X2® to room temperature and vortex gently before using.
Place 250 µl of Opti-MEM® I Reduced-Serum Medium in a sterile tube.
Add 50 nM guide RNA (final concentration). For example, add 2.5 µl of a 50 µM stock. If using 2-part crRNA + tracrRNA, add at a 1:1 molar ratio. For example, add 2.5 µl of each 50 µM stock. incubate at room temperature for 10 minutes to allow the crRNA and tracrRNA to anneal.
Add 2.5 µg plasmid DNA encoding Cas9.
Pipet gently to mix completely.
Add 5 µl TransIT-X2® to the diluted DNA mixture.
Pipet gently to mix completely.
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

Add the TransIT-X2®:DNA:RNA complexes (prepared in Step B) drop-wise to different areas of the wells.
Gently rock the culture vessel back-and-forth and from side-to-side to distribute the TransIT-X2® Reagent:DNA complexes.
Incubate for 24-72 hours. It is not necessary to replace the complete growth medium with fresh medium.

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Brochure: CRISPR/Cas9 Genome Editing
Poster: Optimization of DNA, RNA and RNP Delivery Methods for Efficient CRISPR/Cas9 Mediated Cell Engineering
White Paper: Optimization of DNA, RNA and RNP Delivery Methods for Efficient CRISPR/Cas9 Mediated Cell Engineering

Applications: Genome Editing Using CRISPR/Cas9

CRISPR/Cas9 Genome Editing: pDNA + gRNA Transfection

TransIT-X2® Dynamic Delivery System for Plasmid DNA and Guide RNA Oligonucleotide Delivery

Plasmid DNA and Guide RNA Transfection Protocols

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CRISPR/Cas9 Genome Editing:
pDNA + gRNA Transfection