Applications: Genome Editing Using CRISPR/Cas9

CRISPR/Cas9 Genome Editing:
Cas9/gRNA RNP Delivery

TransIT-X2® Dynamic Delivery System and
Ingenio® Electroporation Solution for Ribonucleoprotein (RNP) Delivery

Purified Cas9 Protein can be combined with guide RNA to form an RNP complex to be delivered to cells for rapid and highly efficient genome editing. Benefits of RNP-based genome editing include:

High Efficiency Delivery – Deliver Cas9/gRNA complexes to multiple cell types, including hard to transfect cells such as immune and stem cells
High Specificity – Pre-formed RNP complexes provide a rapid pulse of genome editing activity
DNA Free – No risk of insertional mutagenesis

RNP Delivery Method | Experimental Data | RNP Transfection Protocol

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CRISPR/Cas9 Delivery Methods – Cas9 RNP. Purified Cas9 protein and guide RNA oligonucleotides are combined to form a ribonucleoprotein (RNP) complex.

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TransIT-X2® Outperforms Lipofectamine® for RNP Delivery. Ribonucleoprotein (RNP) complexes composed of PPIB (cyclophilin B) targeting 2-part gRNA (IDT) and Cas9 protein (PNA Bio) were delivered into HEK293T/17 and U2OS cells using TransIT-X2® Dynamic Delivery System (1 µl/well, Mirus Bio) or Lipofectamine® CRISPRMAX™ (1.5 µl/well and 1 µl/well of Lipofectamine® Cas9 Plus™ Reagent, ThermoFisher) or Lipofectamine® RNAiMAX (1.5 µl/well, ThermoFisher) or Lipofectamine® 3000 (1.5 µl/well and 1 µl/well of P3000™ Reagent, ThermoFisher) in a 24-well format according to the manufacturers’ protocol. Varying levels of gRNA (6 nM or 12 nM) were tested with 6 nM Cas9 protein (PNA Bio). A T7E1 mismatch detection assay was used to measure cleavage efficiency at 48 hours post-transfection.

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Efficient Genome Editing with Cas9 + Guide RNA Ribonucleoprotein Complexes. The RNP complex of PPIB targeting 2-part gRNA (Dharmacon) and Cas9 protein (PNA Bio) was delivered into HEK293T/17, U2OS, NHDF and K562 cells using TransIT-X2® Dynamic Delivery System (1 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. High levels of gene editing can be achieved in cells that were transfected with an RNP complex comprised of 50nM of gRNA and 25nM of Cas9 protein.

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Efficient Genome Editing with Cas9 + Guide RNA Ribonucleoprotein Complexes. TransIT-X2® Dynamic Delivery System was used to deliver Cas9 protein/guide RNA ribonucleoprotein (RNP) complexes in human induced pluripotent stem cells (iPSCs). A T7E1 mismatch assay was used to measure cleavage efficiency at 48 hours post-transfection.

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CRISPR RNP Delivery with Ingenio Electroporation Solution Targeting PPIB & WTAP

CRISPR RNP Delivery with Ingenio® Electroporation Solution. Ribonucleoprotein (RNP) complexes targeting (A) PPIB or (B) WTAP were electroporated into K562 and Jurkat cells. The RNP complex, composed of 750 nM Cas9 protein (EnGen® Cas9 NLS, New England Biolabs) and 1500 nM pre-complexed two-part gRNA (IDT), was electroporated using the Ingenio® Electroporation Solution (Mirus Bio) and a Gene Pulser Xcell™ Eukaryotic System (Bio-Rad® Laboratories). Exponential pulse conditions of 130V (A) & 150V (B), 950 µF for K562 and 150V, 950 µF for Jurkat cells were applied to triplicate 0.2 cm cuvettes, 100 µl volume, 10 x 106 cells/ml +/- RNP complex. A T7E1 mismatch assay was used to measure cleavage efficiency at 48 hours post-transfection. Non-specific bands (NSP) were observed in the negative control of both cell lines. Cleavage efficiency was calculated based on the ratio of cleaved band intensities to the sum of cleaved and uncleaved band intensities minus the average signal of the non-specific band(s) in negative control lanes.

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TransIT-X2® Outperforms Lipofectamine® CRISPRMAX™ for RNP Delivery. Ribonucleoprotein (RNP) complexes composed of PPIB targeting 2-part gRNA (IDT) and Cas9 protein (PNA Bio) were delivered into HEK293T/17 (A), U2OS (B), and primary H-RPE (C) cells using TransIT-X2® Dynamic Delivery System (1 µl/well, Mirus Bio) or Lipofectamine® CRISPRMAX™ (1.5 µl/well and 1 µl/well of Lipofectamine® Cas9 Plus™ Reagent, ThermoFisher) in a 24-well format according to the manufacturers’ protocol. Varying levels of gRNA (6 nM – 60 nM) were tested with 6 nM Cas9 protein. 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.

RNP Delivery Protocol

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CRISPR Ribonucleoprotein Transfection ProtocolClick to expand

CRISPR RNP Transfection Workflow

Ribonucleoprotein (RNP) Transfection Protocol: The following procedure describes how to perform RNP 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®:RNP 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 12 nM guide RNA (final concentration). For example, add 0.6 µl of a 50 µM stock. If using 2-part crRNA + tracrRNA, add at a 1:1 molar ratio. For example, add 0.6 µl of each 50 µM stock. Incubate at room temperature for 10 minutes to allow the crRNA and tracrRNA to anneal.
Add 6 nM purified Cas9 protein (final concentration). For example, add 0.5 µl of a 30 µM stock.
Pipet gently to mix completely. Incubate at room temperature for 10 minutes.
Add 5 µl TransIT-X2® to the diluted RNP mixture.
Pipet gently to mix completely.
Incubate at room temperature for 15-30 minutes to allow sufficient time for complexes to form.

NOTE: We have tested concentrations of Cas9 protein from 6 - 25 nM (final concentration per well) in a variety of cell types. The optimal concentration of Cas9 protein and gRNA per experiment may vary depending on the cell type, the Cas9 protein used, gRNA sequence and delivery method.

C. Distribute the complexes to cells in complete growth medium

Add the TransIT-X2®:RNP 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:RNP 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: Cas9/gRNA RNP Delivery

TransIT-X2® Dynamic Delivery System and Ingenio® Electroporation Solution for Ribonucleoprotein (RNP) Delivery

RNP Delivery Protocol

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CRISPR/Cas9 Genome Editing:
Cas9/gRNA RNP Delivery

TransIT-X2® Dynamic Delivery System and
Ingenio® Electroporation Solution for Ribonucleoprotein (RNP) Delivery