CRISPR Transfection: Efficient Genome Editing

Introduction to Genome Editing

There are various options to modulate gene expression, be it on the DNA, RNA, or protein level. Many of these options only result in a transient modulation that might be sufficient or even advantageous for some approaches. A stable, heritable DNA modification was accomplished either by random integration of plasmids, transposons, or viruses or via homologous recombination. The latter method results in site-specific integration but is a very time-consuming and inefficient process. With the introduction of advanced genome editing tools, site-specific stable modifications can now be performed easily.

Zinc Finger Nucleases (ZFN) and Transcriptional Activator-like Effector Nucleases (TALEN) technologies were established over the last decade as useful tools for site-specific genomic modifications, but with the recent discovery of the CRISPR (clustered regularly interspaced short palindromic repeats) technology another potent alternative has emerged.

Targeted modification of a cell’s DNA via genome editing provides a powerful tool for fundamental biological research, early stage drug discovery, and for the development of novel cellular therapeutics, e.g. immunotherapy.

This technology makes use of engineered nucleases to delete, insert or replace a gene at a targeted genomic location (Gaj T et al., 2013). The nuclease is generating a double-strand break in the genomic DNA and thus inducing one of two possible cellular repair processes. Non-homologous end-joining (NHEJ) can lead to a deletion of the functional gene, since it is often accompanied with mutations when closing the break. If a partially homologous donor sequence is available, an insertion or replacement of a gene can take place via homology-dependent repair (HDR). To perform such a modification at a specific target sequence, the nuclease is fused to or interacts with sequence-specific DNA-binding component directing the nuclease to the target sequence.

Reference:

Gaj T et al (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology 31(7):397-405 (review)

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Genome Editing Tools - Brief Overview

This chapter gives a brief introduction of the process and the main tools used. For more details please refer to the various reviews available (e.g. Gaj T et.al., 2013).

As mentioned above, for genome editing, engineered nucleases are used to delete, insert, or replace a gene at a targeted genomic location. Such engineered nucleases are typically comprised of two elements: an endonuclease DNA cleavage module, and a sequence-specific DNA binding domain. The nuclease cleaves double-stranded DNA creating a double-strand break (DSB). The DSB induces the cellular DNA repair process. There are two types of repair processes that can occur. Without a homologous donor fragment available – be it the corresponding allele or an external donor DNA – the broken ends will be re-joined. This process is called non-homologous end joining (NHEJ) and is often accompanied by a mutation that may cause a deletion of a functional element of the gene.

If a partially homologous donor sequence is present, e.g. the genomic allele or foreign donor DNA, an insertion or replacement of a gene can take place via homology-dependent repair (HDR). The frequency of NHEJ versus HDR depends on the individual experimental setting, e.g. the cell-type and the donor amount.

The combination of such nucleases with a sequence-specific DNA binding domain that can be customized to recognize virtually any sequence facilitates these repair processes in a targeted manner.

The typically used DNA binding domains for site-specific genome editing tools are:

ZFN (Zinc Finger Nucleases)
TALEN (Transcriptional Activator-Like Effector Nucleases)
CRISPR/Cas9 (Clustered Regularly Interspaced Palindromic Repeats and CRISPR-Associated nuclease 9)

Cellular repair processes following the nuclease-induced double-strand break (simplified scheme)

Nuclease

Cas9

Fok1

DNA binding molecule

GuideRNA (gRNA)

ZF Protein

TALE Protein

Type

Protein + RNA
→ Easy to modify
→ Multiple targeting possible

Fusion protein
→ High effort to modify for new targeting site

Binding sites

1 site (18-20 bp + 3bp PAM)
→ Lower specificity
→ Higher risk for off-target effects

2 sites (15 or 18 bp each)
→ High specificity
→ Low risk for off-target effects

2 sites (≥ 13 bp each)
→ High specificity
→ Low risk for off-target effects

Reference: Gaj T et al (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology 31(7):397-405 (review)

CRISPR/Cas9 System

The CRISPR (clustered regularly interspaced palindromic repeats) technology is based on a bacterial defense pathway against viral invasion. It has been adapted for genome editing applications in eukaryotic cells and is an emerging technology because it allows for some more flexibility compared to zinc finger nucleases (ZFNs) or transcriptional activator-like effector nucleases (TALENs).

For further details, see our Technical Reference Guide, "Genome Editing using Nucleofector^TM Technology".

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Genome Editing using Nucleofector™ Technology

Technical Reference Guide providing a brief introduction to genome editing tools and technical tips for ZFN, TALEN or CRISPR delivery using the Nucleofector™ Technology

In contrast to ZFN and TALEN (see below for more information), CRISPR-based genome editing is relying on two separate components working together:

The DNA targeting part is the so called guideRNA (gRNA) that can be designed as such that it is complimentary to an 18-21 bp stretch of the genomic DNA.
Once paired with the genomic DNA strand, it recruits Cas9 nuclease (CRISPR-associated nuclease 9) to the genomic DNA which introduces a double-strand break at a defined site.

Since the DNA-specific part is an RNA molecule it is much easier to design and produce than a ZF- or TALE-nuclease fusion protein. In addition, by transferring Cas9 nuclease together with several gRNAs targeting different sites multiple genome editing is possible.

Reference:

Gaj T et al (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology 31(7):397-405 (review)

CRISPR-based Genome Editing Requires Co-transfection

For CRISPR transfection, various scenarios are possible:

Transfer of one plasmid carrying both the gRNA and Cas9 nuclease
Co-transfection of two separate plasmids (one for the gRNA and one for Cas9)
Co-transfection of a plasmid carrying Cas9 and a PCR cassette expressing the gRNA
A Cas9-gRNA ribonucleoprotein (RNP) complex
When aiming for insertion or replacement, for all scenarios an additional donor/repair plasmid or a single strand oligonucleotide (ssODN) has to be co-transfected

Our Nucleofector^TM Technology has been shown to work as a reliable transfection method for CRISPR-based genome editing tools

High transfection efficiencies for a broad range of cell types, including iPSCs
Efficient co-transfection of various substrates
Same conditions for transfecting plasmids, DNA, mRNA or PCR cassettes, ssODN

The Nature Protocols publication from Ran et al. (2013) provides a very comprehensive guideline on using the 4D-Nucleofector^TM System for CRISPR transfection.

Selected references for CRISPR transfection:

Petit CS et al. (2013) J Cell Biol 202:1107-1122 (cells: HeLa)

Ran FA et al. (2013) Cell 154:1380–1389 (cells: various cell lines, e.g. HEK293FT)

Ran FA et al. (2013) Nat Prot 8(11):2281–2308 (cells: HEK293 and HUES62)

Seki and Rutz (2018) J Exp Med 215(3):985-997

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Genome Editing of Immune Cells

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Efficient CRISPR/Cas9 Delivery Using Nucleofector^{^®} Technology

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For further technical assistance on using the Nucleofector^{^®} Technology for CRISPR-based genome editing, contact Lonza Scientific Support.

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Genome Editing Publications List

Genome editing publications list providing an overview about tools (including cell types and substrate types) that were successfully transfected using Nucleofector^TM Technology

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Zinc Finger Nucleases (ZFN)

Zinc finger (ZF) proteins are the most abundant and versatile DNA binding motif in nature (Tupler R et al. (2001). An individual zinc finger domain binds 3 DNA base pairs. Because of their modular structure, they provide an ideal framework for designing an artificial sequence-specific binding molecule. Fusion of a ZF to an endonuclease (mostly Fok1) builds a zinc finger nuclease (ZFN). Two such ZFNs work in combination to bind the sense and antisense strand of the targeted DNA sequence. After binding, the Fok1 nuclease forms an active dimer and induces the double-strand break that leads to subsequent cellular repair processes.

The Nucleofector^TM Technology has been successfully used for ZFN transfection.

For technical guidelines please, download our Technical Reference Guide.

References:

Tupler R et al. (2001) Nature 409: 832-833

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Transcriptional Activator-Like Effector Nucleases (TALEN)

In 2009 transcriptional activator-like effectors (TALEs) were discovered to provide a simpler, modular DNA recognition code (Boch et al., 2009; Moscou et al., 2009). TALEs are naturally occurring proteins from the plant pathogenic bacteria genus Xanthomonas, and contain DNA-binding repeats, each recognizing a single base pair. Compared to the triplet-based DNA binding of zinc fingers, this single base recognition mode of TALE–DNA binding repeats enables greater flexibility in design but also holds some cloning challenges. Thus, except for the binding mode, the principle of targeting is very similar. Again sequence-specific, engineered TALEs are typically fused to Fok1 nuclease to build the TALE-nuclease fusion (TALEN). As with ZFNs, a pair of TALENs must be generated for each target with each monomer binding (Jinek et al., 2013) or more base pairs on the sense or antisense strand of the targeted DNA.

The Nucleofector^TM Technology has been successfully used for TALEN transfection.

For technical guidelines, please download our Technical Reference Guide.

References:
Boch J et al. (2009) Science 326 (5959): 1509–12
Jinek M et al. (2013) eLife 2: e00471
Moscou MJ et al. (2009) Science 326 (5959): 1501
Ran FA et al. (2013) Nat Prot 8(11):2281–2308 (cells: HEK293 and HUES62)

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Validating and Scaling CRISPR Applications

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CRISPR – Tips and Tricks for Efficient Delivery into Primary Cells and Cell Lines

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Over the last few years, CRISPR/Cas9 has emerged as the genome editing method of choice. Explore the full CRISPR potential and hear about best practices to achieve optimal delivery results.

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CRISPR/Cas9 Cell Therapeutics – The Next Generation of Cures

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CRISPR Transfection: Efficient Genome Editing using NucleofectorTM Technology

Introduction to Genome Editing

Request a Demo - Online or In Person

Nucleofector™ Technology

Genome Editing Tools - Brief Overview

Cellular repair processes following the nuclease-induced double-strand break (simplified scheme)

Genome Editing using Nucleofector™ Technology

CRISPR-based Genome Editing Requires Co-transfection

Genome Editing of Immune Cells

Efficient CRISPR/Cas9 Delivery Using Nucleofector® Technology

Genome Editing Publications List

Validating and Scaling CRISPR Applications

CRISPR – Tips and Tricks for Efficient Delivery into Primary Cells and Cell Lines

CRISPR/Cas9 Cell Therapeutics – The Next Generation of Cures

Discover the Next Scale

Interested in our Webinars?

Subscribe to our eNewsletter

CRISPR Transfection: Efficient Genome Editing using Nucleofector^TM Technology

Efficient CRISPR/Cas9 Delivery Using Nucleofector^{^®} Technology