Applications | Genome Editing Using CRISPR/Cas9

Transfection Methods for CRISPR/Cas9 Genome Editing

TRANSFECTION TIPS VIDEO: CRISPR/Cas9 Transfection This video covers the basic mechanism of CRISPR/Cas9 genome editing and introduces the key points affecting researchers’ decisions about whether to deliver Cas9 and guide RNA in plasmid, RNA or RNP format.

Overview

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. Target cleavage by Cas9 requires base-pairing between the crRNA and tracrRNA as well as base pairing between the crRNA and the target DNA (See figure CRISPR/Cas9 Genome Editing). Target recognition is facilitated by the presence of a short sequence called a protospacer-adjacent motif (PAM) that conforms to the sequence NGG.

Editing Mammalian Genomes with CRISPR

The bacterial CRISPR/Cas9 system has been adapted to serve as a versatile platform for RNA-directed genome editing in mammalian cells. The Cas9 endonuclease can be programed by a dual RNA (crRNA and tracrRNA), or the core components of these RNAs can also be combined into a single hybrid guide RNA. Once the Cas9 has cleaved the target DNA, 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.

Advantages

The simplicity of using of a noncoding RNA guide to target DNA for site-specific cleavage provides a distinct advantage over alternative genome editing technologies such as ZFNs and TALENs. Using the CRISPR/Cas9 strategy, retargeting the nuclease complex only requires introduction of a new RNA sequence and there is no need to reengineer the specificity of DNA-binding proteins.
CRISPR Cas9 genome editing- Illustration
CRISPR/Cas9 Genome Editing. The Cas9 endonuclease (blue) is targeted to DNA by a guide RNA which can be supplied as a two-part system consisting of crRNA and tracrRNA or as a single guide RNA, where the crRNA and tracrRNA are connected by a linker (dotted line). Target recognition is facilitated by the protospacer-adjacent motif (PAM). Cleavage occurs on both strands (scissors) 3 bp upstream of the PAM.
CRISPR Handles Multiple Types of Genome Modification Illustration
Multiple Genomic Alterations are Possible Following Cleavage of Target DNA by Cas9. Variable length insertions and/or deletions (indels) can result near the DNA break due to mistakes in DNA repair by the endogenous non-homologous end joining (NHEJ) pathway. These indels frequently result in disruption of gene function. Alternatively, by supplying a DNA repair template, researchers can leverage the homology-directed repair (HDR) pathway to create defined deletions, insertions or modifications.
CRISPR Multiple Delivery Methods Summary Data
DNA, RNA and Protein Formats for CRISPR Genome Editing. TransIT-X2® Dynamic Delivery System was used to deliver Cas9 pDNA/gRNA and Cas9 protein/gRNA (RNP complex). TransIT®-mRNA was used to deliver Cas9 mRNA/gRNA.  A T7E1 mismatch assay was used to measure cleavage efficiency at 48 hours post-transfection. For more details, see our methods for delivery of CRISPR/Cas9 DNA, RNA and RNP.
CRISPR Delivery Methods Pros and Cons Comparison
Pros and Cons of DNA, RNA and Protein Formats for Genome Editing. Cas9 can be delivered as plasmid DNA for a simple, low-cost approach. Cas9 mRNA enables rapid gene expression, and eliminates the risk of insertional mutagenesis. Cas9/guide RNA ribonucleoprotein (RNP) complexes exhibit the most rapid pulse of genome editing activity and reduce the possibility of off-target cleavage events. Cas9 mRNA and RNP formats can also be efficiently delivered to cell types that are resistant to transfection with plasmid DNA.

Glossary of CRISPR Terms

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.

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
CRISPR Plasmid Delivery Approaches Illustration

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.

 
CRISPR Plasmid and gRNA Delivery Approaches Illustration

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.

 
CRISPR Plasmid and gRNA Delivery Data

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 Terms

TermDefinition
Cas9CRISPR 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 NickaseA 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.
CRISPRClustered 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.
crRNACRISPR 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.
DSBDouble 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 EditingThe creation of desired genetic modifications including gene insertions, deletions, or replacements through the use of targeted nucleases such as Cas9.
gRNAGuide 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).
HDRHomology 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 AssayA 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.
NHEJNon-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 EffectsOff-target effects refers to Cas9 cleavage events at genomic locations other than the intended site.
PAMProtospacer-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.
sgRNASingle Guide RNA, a chimeric RNA composed of crRNA and tracrRNA, connected by a short RNA linker.
Target SequenceA 20 nucleotide genomic DNA sequence which base-pairs with gRNA and is cleaved by Cas9.
tracrRNATrans-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

Download DNA Transfection Protocol (PDF)
Download DNA/RNA Transfection Protocol (PDF)

 

CRISPR pDNA Transfection Protocol

CRISPR Cas9 pDNA Transfection Workflow Schematic

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

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 (PDF).

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 encoding Cas9 and/or guide RNA.
  4. Pipet gently to mix completely.
  5. Add 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.

 

CRISPR pDNA + gRNA Transfection Protocol

CRISPR Cas9 pDNA gRNA Transfection Workflow Schematic

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

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 (PDF).

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:RNA 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 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.
  4. Add 2.5 µg plasmid DNA encoding Cas9.
  5. Pipet gently to mix completely.
  6. Add 5 µl TransIT-X2® to the diluted DNA mixture.
  7. Pipet gently to mix completely.
  8. 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:RNA 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.

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
 
CRISPR mRNA and gRNA Delivery Approaches Illustration

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.

 
CRISPR mRNA and gRNA Delivery Data

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 Terms

TermDefinition
Cas9CRISPR 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 NickaseA 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.
CRISPRClustered 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.
crRNACRISPR 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.
DSBDouble 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 EditingThe creation of desired genetic modifications including gene insertions, deletions, or replacements through the use of targeted nucleases such as Cas9.
gRNAGuide 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).
HDRHomology 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 AssayA 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.
NHEJNon-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 EffectsOff-target effects refers to Cas9 cleavage events at genomic locations other than the intended site.
PAMProtospacer-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.
sgRNASingle Guide RNA, a chimeric RNA composed of crRNA and tracrRNA, connected by a short RNA linker.
Target SequenceA 20 nucleotide genomic DNA sequence which base-pairs with gRNA and is cleaved by Cas9.
tracrRNATrans-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.
 

CRISPR mRNA + gRNA Transfection

CRISPR Cas9 mRNA Transfection Workflow Schematic

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 (PDF).

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 transfection complexes (Immediately before transfection)

  1. Warm TransIT®-mRNA and mRNA Boost 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 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.
  4. Add 2.5 µg mRNA encoding Cas9.
  5. Pipet gently to mix completely.
  6. Add 2.5 µl mRNA Boost Reagent to the diluted RNA mixture.
  7. Pipet gently to mix completely.
  8. Add 2.5 µl TransIT®-mRNA Reagent.
  9. Pipet gently to mix completely.
  10. 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

  1. Add the transfection 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 transfection complexes.
  3. Incubate for 24-72 hours. It is not necessary to replace the complete growth medium with fresh medium.

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
 
CRISPR RNP Delivery Approach Illustration

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

 
TransIT-X2 Dynamic Delivery System vs Lipofectamine 3000 vs CRISPRMAX Data

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.

 
CRISPR RNP Delivery Data

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.

 
CRISPR RNP Delivery Data iPSC Data

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.

 
Ingenio Electroporation Solution CRISPR RNP Delivery PPIB WTAP Data

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.

 
TransIT-X2 Dynamic Delivery System vs CRISPRMAX Data

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 Terms

TermDefinition
Cas9CRISPR 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 NickaseA 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.
CRISPRClustered 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.
crRNACRISPR 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.
DSBDouble 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 EditingThe creation of desired genetic modifications including gene insertions, deletions, or replacements through the use of targeted nucleases such as Cas9.
gRNAGuide 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).
HDRHomology 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 AssayA 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.
NHEJNon-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 EffectsOff-target effects refers to Cas9 cleavage events at genomic locations other than the intended site.
PAMProtospacer-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.
sgRNASingle Guide RNA, a chimeric RNA composed of crRNA and tracrRNA, connected by a short RNA linker.
Target SequenceA 20 nucleotide genomic DNA sequence which base-pairs with gRNA and is cleaved by Cas9.
tracrRNATrans-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 Protocols

Download RNP Transfection Protocol (PDF)

Download RNP + DNA Oligo Transfection Protocol (PDF)

Download RNP Electroporation Protocol (PDF)

 

CRISPR Ribonucleoprotein Transfection Protocol

CRISPR Cas9 RNP Transfection Workflow Schematic

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 (PDF).

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®:RNP 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 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.
  4. Add 6 nM purified Cas9 protein (final concentration). For example, add 0.5 µl of a 30 µM stock.
  5. Pipet gently to mix completely. Incubate at room temperature for 10 minutes.
  6. Add 5 µl TransIT-X2® to the diluted RNP mixture.
  7. Pipet gently to mix completely.
  8. 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

  1. Add the TransIT-X2®:RNP 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:RNP complexes.
  3. Incubate for 24-72 hours. It is not necessary to replace the complete growth medium with fresh medium.

Products for Genome Editing Research

TransIT-X2 Dynamic Delivery System Sample

TransIT-X2® Dynamic Delivery System

A novel, polymeric system for delivery of multiple nucleic acids to mammalian cells. Delivers CRISPR/Cas9 components in the following formats:

  • DNA – Deliver plasmid DNA expressing Cas9 or guide RNA
  • Guide RNA – Deliver guide RNA oligonucleotides targeting your gene of interest
  • RNP – Deliver Cas9/guide RNA ribonucleoprotein complexes

TransIT-mRNA Transfection Reagent Sample

TransIT®-mRNA Transfection Kit

A high efficiency, low toxicity transfection reagent for mammalian cells. Delivers CRISPR/Cas9 components in the following formats:

  • mRNA – Deliver messenger RNA expressing Cas9
  • Guide RNA – Deliver guide RNA targeting your gene of interest

IngenioElectroporation Solution Sample

Ingenio® Electroporation Solution

A high efficiency electroporation solution compatible with most conventional electroporation devices including Lonza-Amaxa®, Bio-Rad® or Harvard BTX®. Delivers CRISPR/Cas9 components in the following formats:

  • DNA – Deliver plasmid DNA expressing Cas9 or guide RNA
  • mRNA – Deliver messenger RNA expressing Cas9
  • Guide RNA – Deliver guide RNA oligonucleotides targeting your gene of interest
  • RNP – Deliver Cas9/guide RNA ribonucleoprotein complexes
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Technical Resources

Don’t See Your Cell Type? Consult Reagent Agent® Transfection Database
Citation Database: Check if our reagents have been used by other researchers to transfect your cell type
Technical Support: Communicate directly with a transfection expert

Poster: Optimization of DNA, RNA and RNP Delivery Methods for Efficient CRISPR/Cas9 Mediated Cell Engineering (PDF)
White Paper: Optimization of DNA, RNA and RNP Delivery Methods for Efficient CRISPR/Cas9 Mediated Cell Engineering (PDF)

"I was recently tasked with developing a CRISPR protocol for primary and bone-derived cell lines. TransIT-X2® was simple to use, 2-3 times better for transfection and much gentler on my cells than other products! I feel I have hit the jackpot and have already passed this exciting information on to my colleagues."
Joshua Chou, Ph.D.
Harvard School of Dental Medicine