June 15, 2015 (Vol. 35, No. 12)

Ellen Prediger, Ph.D. IDT

Developing cell lines for use as accurate models for disease has always been a challenging task. In order to replicate phenotypes or enable downstream experimentation, genome engineering of cell lines is required. Techniques enabling genome modification have evolved over a number of years, from simple viral vectors used to insert sequences in random locations, to more sophisticated methods that enable targeting of specific loci. The ability to edit genes in human cells in a highly targeted manner has been transformative, expanding our capacity to understand human genetics and providing the potential for therapeutic applications.

CRISPR/Cas9 has recently emerged as a flexible, accurate, and cost-effective system for genome modification that has rapidly increased in popularity over the past two years due to several advantages it holds over other methods. Recognizing the vast potential of this technique in creating disease models, researchers are busy developing new methods to build upon its strengths. Here we describe protocols that alleviate some of the practical issues still faced in using CRISPR/Cas9, particularly in high-throughput applications.


Genome Editing and CRISPR

Inserting or deleting DNA sequences within the genome requires at least one of the two strands to be cleaved, activating either the “non-homologous end joining” or the “homologous recombination” mechanism of DNA repair. During the repair process, new DNA sequences can be inserted at the break site. A number of methods exist to take advantage of this natural system, including Zinc-finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). But most of these methods suffer from time-consuming protocols involving the synthesis of target-specific proteins. As a result, set up can be costly and reliability variable.

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) and the Cas9 endonuclease were originally identified in bacteria and archaea as a naturally occurring, adaptive defense mechanism, and researchers were quick to recognize its potential for genome editing. Since 2012, the technique has been refined, improved upon, and modified to expand its range of uses.


Improving CRISPR/Cas9 Targeting

One of the two major components of this system, Cas9, is typically expressed on a transfectable plasmid, although a cassette can instead be integrated into the genome. The other target-specific component is known as the single guide RNA (sgRNA). This is a chimera that contains the 20 base target specific sequence and the Cas9-binding sequence. The sgRNA is often cloned into and expressed from the same plasmid vector as Cas9, but under expression control by a different promoter. The sgRNA may also be transfected separately, or, alternatively, both Cas9 and sgRNA can be in vitro transcribed (IVT) and microinjected into individual cells (Figure 1). Once they create a double-strand break in the targeted genomic sequence, new DNA can be inserted.

One of the early modifications made to this system by the Church and Zhang labs at Harvard and MIT, respectively, was to enhance specificity. This involved disabling one endonuclease site in Cas9, thereby enabling single-strand nicking. Using this variation of the system for genome editing requires two independent sgRNAs for targeting, and this dual specificity was shown to increase targeting efficiency, and therefore accuracy, as much as 100-fold.


Figure 1. Popular CRISPR approaches: Combined Cas9/sgRNA plasmid vectors have been the most popular approach to CRISPR-mediated gene editing. Alternatives include separate transfection of sgRNA constructs and in vitro transcription of both Cas9 and sgRNA constructs, followed by microinjection. All methods enable highly targeted gene editing through homologous recombination (HR) or non-homologous end joining (NHEJ).

CRISPR for High-Throughput Applications

While a number of further modifications have expanded the system’s usability, significant issues remain for researchers—transfection efficiency and accurate assessment of gene editing efficiency. Successful transfection can be directly affected by vector size, with a noticeable reduction in efficiency usually seen as size increases. Large plasmids (>9 kb), such as the often used Cas9/sgRNA combined plasmid, can therefore significantly reduce efficiency. Furthermore, generating these plasmid constructs for each individual target can often be a laborious task.

As stated above, IVT RNAs can serve as the sgRNA but are also not amenable to high-throughput applications. IVTs also have the added risk of immune stimulation and cell death. It is important to verify complete removal of the triphosphate to reduce this risk.

Providing a solution to these issues, here we describe an optimized protocol that was recently published by the Church lab. Particularly useful for high-throughput applications, it includes the synthesis and direct transfection (without cloning into the Cas9 vector) of sgRNA constructs using Integrated DNA Technologies (IDT) gBlocks® Gene Fragments. This allows a smaller Cas9 vector to be used for transfection and inexpensive, rapid screening of many sgRNA sequences.

Optimized Transfection
In order to simplify the cloning and transfection steps, a standard human Cas9 plasmid vector (AddGene #418815) is used. This version of Cas9 has two active endonuclease sites, enabling the creation of a double-strand break. A Cas9-integrated cell line may be used as an alternative where appropriate.

For targeting, sequence analysis software is utilized to identify 22 bp regions in proximity to the intended target in the form of 5´-N19-NGG-3´. The candidate sequences are then used to check for alternative binding sites in the reference genome. The most suitable candidate sequence is expanded to include a U6 promoter, sgRNA, and transcription terminator. This final 455 bp expression fragment is then synthesized as an inexpensive, standard IDT gBlocks Gene Fragment, thereby removing any further cloning steps.

This fragment can be amplified by either blunt cloning into a suitable vector, such as pCRII-Blunt-TOPO, or simply by high fidelity PCR. It is then directly transfected as a PCR-amplified expression fragment or a plasmid, though higher efficiencies may be achieved by directly transfecting the expression fragment.

Transfection into human iPSCs or HEK293 cells is conducted using established electroporation (for human iPSCs) or Lipofection-based (for HEK293) methods.

Assessing Gene Editing Efficiency
Gene editing efficiency may vary depending on the methodology, and it is vital to assess performance. It may be quickly and quantitatively assessed as follows.

Firstly, genomic DNA is isolated from both transfected and control cells, and amplified across the region of interest by high fidelity PCR. 300 ng of each PCR product is then heat denatured and allowed to form heteroduplexes from the wild type and edited DNA present as the sample cools. A suitable mismatch endonuclease is then used to cleave the fragments. The ratio of full size to cleaved fragments indicates the editing efficiency, when analyzing these digest products via capillary gel electrophoresis.

The Fragment Analyzer™ (AATI) is an automated capillary gel electrophoresis system that allows for rapid visualization and reliable quantification of many digestion reactions.

Figure 2 illustrates how this method is employed to compare the editing efficiency of gBlocks Gene Fragments and cloned sgRNA plasmid vectors in HEK293-Cas9 cells. In testing multiple sgRNAs targeting human HPRT, the editing efficiency is found to be comparable. This illustrates that, without the need for additional cloning steps, efficiencies matching or exceeding plasmid vectors are achieved by direct transfection of gBlocks Gene Fragments, making them ideal for high-throughput applications.


Figure 2. Quantitative assessment of gene editing efficiency using the mismatch endonuclease technique. Time course and dose response study using sgRNA constructs (gBlocks® Gene Fragments) in HEK293-Cas9 cells shows optimal cleavage efficiency with 3 nM gBlocks after 48 hours (A). Comparison of editing efficiency in HEK293-Cas9 cells using 10 sgRNA variants targeting Hs HPRT transfected in as plasmid vectors (100 ng) or gBlocks Gene Fragments (3 nM final). Editing efficiency is comparable or higher with gBlocks Gene Fragments (B). Percent cleavage values were determined via analysis on the Fragment Analyzer.

Conclusions

Enabling simpler and more cost-effective solutions to both performing and assessing CRISPR/Cas9-mediated gene editing in human cell lines, the methods described here have been developed to tackle practical issues faced by researchers. 

The use of a fully synthesized sgRNA transcription fragment reduces the number of protocol steps, and therefore hands-on time required to perform gene editing. This approach is rapidly increasing in popularity, with >14,000 different sgRNA constructs made as IDT gBlocks Gene Fragments to date. The direct transfection of these sgRNA constructs into cell lines, as described, results in high editing efficiencies that are comparable to using sgRNA plasmid vectors, making these inexpensive constructs especially useful where high-throughput is required. In addition, the mismatch endonuclease method described here provides a rapid and quantitative method of assessing gene editing efficiency.

These methods will enable researchers to make better use of their time and resources, ensuring CRISPR/Cas9-mediated gene editing remains at the cutting edge of genome engineering.



























Ellen Prediger, Ph.D. (eprediger@idtdna.com), is a senior writer at Integrated DNA Technologies (IDT).  For more information on these methods and the CRISPR/Cas9 system, see www.idtdna.com/CRISPR.

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