Applications: Genome Editing Using CRISPR/Cas9

CRISPR/Cas9 Genome Editing:
mRNA + gRNA Transfection

TransIT®-mRNA Transfection Kit for mRNA and gRNA Oligonucleotide Delivery

In order to avoid off-target cleavage and unwanted genomic integration of plasmid DNA, Cas9-encoding mRNA can be co-transfected with guide RNA oligonucleotides. Benefits of RNA-based genome editing include:

High Specificity Rapid gene expression generates a transient pulse of genome editing activity
Ease of Use Deliver mRNA and guide RNA with a single reagent
DNA Free No risk of insertional mutagenesis

RNA Transfection Protocol

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CRISPR mRNA and gRNA Delivery Approaches

CRISPR/Cas9 Delivery Methods – Cas9 mRNA + Guide RNA Oligonucleotides. Cas9 is supplied as messenger RNA, and guide RNAs 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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Delivery of Cas9 mRNA and CRISPR Guide RNA with TransIT-mRNA Transfection Kit

Efficient Genome Editing with Cas9 mRNA + Guide RNA Oligonucleotides. HEK293T/17, U2OS and NHDF cells were co-transfected with 0.5 µg of Cas9 encoding mRNA, 5meC, ψ (Trilink Biotechnologies) and 25nM of PPIB targeting 2-part gRNA (Dharmacon) using TransIT®-mRNA Transfection Kit (0.5 µl/well of 24-well plate of both mRNA Reagent and Boost, 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.

RNA Transfection Protocol
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CRISPR mRNA + gRNA Transfection ProtocolClick to expand

CRISPR mRNA Transfection Workflow

RNA Transfection Protocol: The following procedure describes how to perform messenger RNA and guide RNA co-transfections using TransIT®-mRNA Transfection Kit in 6-well plates. If using vessels with different surface areas, scale accordingly. For more details on performing transfections with TransIT®-mRNA, 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 transfection complexes (Immediately before transfection)

Warm TransIT®-mRNA and mRNA Boost 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 mRNA encoding Cas9.
Pipet gently to mix completely.
Add 2.5 µl mRNA Boost Reagent to the diluted RNA mixture.
Pipet gently to mix completely.
Add 2.5 µl TransIT®-mRNA Reagent.
Pipet gently to mix completely.
Incubate at room temperature for 2-5 minutes to allow sufficient time for complexes to form.

C. Distribute the complexes to cells in complete growth medium

Add the transfection 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 transfection 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: mRNA + gRNA Transfection

TransIT®-mRNA Transfection Kit for mRNA and gRNA Oligonucleotide Delivery

RNA Transfection Protocol DOWNLOAD RNA TRANSFECTION PROTOCOL

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CRISPR/Cas9 Genome Editing:
mRNA + gRNA Transfection

RNA Transfection Protocol
DOWNLOAD RNA TRANSFECTION PROTOCOL