Genome engineering using Cas9/gRNA Ribonucleoproteins (RNPs)

Posted by Joel McDade on Apr 21, 2016 10:30:00 AM


CRISPR has quickly become the preferred system for genome engineering due to its simplicity, as it requires only Cas9 and a guide RNA (gRNA).  Choosing the correct method to deliver both Cas9 and gRNAs to your target cells is absolutely critical as failure to adequately express either component will result in a failed experiment.  In our previous blog post entitled “CRISPR 101 - Mammalian Expression Systems and Delivery Methods” we provided a general overview of the most common ways in which you can deliver Cas9 and gRNAs to your target cells and discussed a few key advantages and disadvantages of each method. In this blog post, we will go into greater detail about why and how Cas9/gRNA Ribonucleoprotein complexes (Cas9 RNPs) are being used for genome engineering experiments and provide a general framework for getting started with Cas9 RNPs in your research.

What Are Cas9 RNPs and Why Are They So Useful?

2016_4_20_CRISPRRNPsFigure1Caption_JoelMcDade.png

Find Validated gRNAs to Use in Your RNP Experiments

Cas9 RNPs consist of purified Cas9 protein in complex with a gRNA.  They are assembled in vitro and can be delivered directly to cells using standard electroporation or transfection techniques.  Cas9 RNPs are capable of cleaving genomic targets with similar efficiency as compared to plasmid-based expression of Cas9/gRNA and can be used for most of the current genome engineering applications of CRISPR, including: generating single or multi-gene knockouts in a wide variety of cell types, gene editing using homology directed repair (HDR), and generating large genomic deletions.

Cas9 RNPs differ from plasmid or viral-based delivery of CRISPR components with regards to how quickly the components are expressed and how long they are present within the cell. Plasmid or viral delivery of Cas9 and gRNA(s) requires the use of cellular transcription/translation machinery to generate functional Cas9-gRNA complexes, which results in a significant lag in peak Cas9 protein expression (>12 hours).  Expression of each component continues indefinitely (for lentiviral-mediated delivery) or until the DNA is lost through cell division (for plasmid or AAV-based delivery).  By contrast, Cas9 RNPs are delivered as intact complexes, are detectable at high levels shortly after transfection, and are quickly cleared from the cell via protein degradation pathways. There are two major consequences of the distinct kinetic profile of Cas9 RNPs.  First, using Cas9 RNPs may increase the rate at which mutations form in target genes compared to plasmid-mediated delivery of Cas9 and gRNAs; Cas9 RNPs are delivered as functional complexes capable of cleaving target DNA and don’t need to be transcribed and translated.  Second, rapid clearance of Cas9 RNPs from the cell may increase CRISPR specificity by reducing the amount of time that Cas9 is available for off-target cleavage.  The aforementioned characteristics of Cas9 RNPs make them useful for CRISPR applications where limited expression of Cas9 is required and specificity is a concern, such as knockout generation or homologous recombination.  Experiments that require long-term expression of Cas9, such as visualizing genomic loci using fluorophore tagged dCas9 may require the use of plasmid or viral-mediated delivery.

You've Decided to Use RNPs, Now What?

The following is a general workflow for using Cas9-RNPs for genome engineering.  First, you will need to obtain purified Cas9 and purified gRNAs targeting your specific locus of interest.   gRNAs should be designed based on standard gRNA design principles, making sure to pick targeting sequences that are upstream of a PAM sequence and unique to the target compared to the rest of the genome.  Purified gRNAs can be generated by PCR amplification of annealed gRNA oligos or in vitro transcription of a linearized gRNA containing plasmid (such as Addgene plasmid 42250 from Keith Joung’s lab).  Cas9 (or a variant of Cas9) can be purified from bacteria through the use of bacterial Cas9 expression plasmids, including these plasmids from Jacob Corn’s group at the Innovative Genomics Initiative.  In most cases, His-tagged Cas9 is expressed in bacterial cells and then purified using nickel affinity chromatography.  Alternatively, purified Cas9 can be purchased from a variety commercial sources including NEB and Thermo Fisher.

Cas9 RNP delivery to target cells is typically carried out via lipid-mediated transfection or electroporation. Liang et al. 2015 compared electroporation to lipid based transfection of Cas9 RNPs for two DNA targets across 11 cell lines.  For several of the cell lines, electroporation yielded high cleavage efficiency when lipid-based delivery completely failed, suggesting that electroporation may be more suitable for difficult to transfect cell types.  Interestingly, the cell types that were resistant to lipid-mediated Cas9 RNP delivery were also resistant to lipid-mediated plasmid delivery.  So, if it is difficult to deliver plasmids to your specific cell type using lipid reagents, it may be difficult to deliver RNPs, as well.  However, Zuris et al. 2014 demonstrated that lipid-mediated delivery can be used to modify genomic targets in human cells in culture and mouse outer hair cells in vivo and recent advances in lipid chemistry may increase the efficiency of lipid-mediated Cas9 RNP delivery. Ultimately, selecting a delivery method for Cas9 RNPs will require some experimentation and optimization for your specific cell type.  Once you have treated your cells with Cas9 RNPs, you should validate your edit, either by isolating individual clones and screening your target locus with Sanger sequencing or analyzing cleavage efficiency using a restriction digest-based assay (T7 endonuclease assay or Surveyor assay).

Wrapping It All Up

In summary, there are several advantages to using Cas9 RNPs in your CRISPR experiment.  Cas9 RNPs can be generated quickly and delivered directly to cells as fully functional Cas9-gRNA complexes.  Cas9 RNPs remove the necessity of cloning targeting oligos into a plasmid backbone, which enables researchers to go from designing gRNA(s) to validating a genome edit in as little as 3-4 days.  Cas9 RNPs are active immediately following transfection and are quickly degraded within the cell. These fast degradation kinetics enable Cas9 RNPs to modify target genes with reduced off-target effects.  Of course, Cas9 RNPs are not without limitations.  A major drawback of using Cas9 RNPs is that expression is transient.  Therefore, it may be best to use plasmid-based delivery or lentiviral-mediated delivery of CRISPR components in cases where stable or elevated expression of CRISPR components is necessary.


References

1. Liang, Xiquan, et al. "Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection." Journal of biotechnology 208 (2015): 44-53. PubMed PMID: 26003884.

2. Zuris, John A., et al. "Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo." Nature biotechnology 33.1 (2015): 73-80. PubMed PMID: 25357182. PubMed Central PMCID: PMC4289409.

3. Kim, Sojung, et al. "Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins." Genome research 24.6 (2014): 1012-1019. PubMed PMID: 24696461. PubMed Central PMCID: PMC4032847.

4. Wang, Ming, et al. "Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles." Proceedings of the National Academy of Sciences 113.11 (2016): 2868-2873. PubMed PMID: 26929348.

Additional Resources on the Addgene Blog

 Resources on Addgene.org

Click to subscribe to Addgene's genome engineering blog posts

Topics: Genome Engineering, CRISPR, Techniques

Blog Logo Vertical.png
Click here to subscribe to the Addgene Blog
 
Subscribe

 

Recent Posts