May 1, 2015 (Vol. 35, No. 9)

You Can Be “On Target” and Still Fail to Win a Prize

The research community’s rapid acceptance of the CRISPR/Cas technology is propelling a stage of deep investment in technology development. Already, three companies have emerged focusing on CRISPR therapeutic applications: Intellia Therapeutics, Editas Medicine, and CRISPR Therapeutics.

To continue to move the technology forward, scientists recently converged at the CRISPR Precision Gene Editing Congress to discuss unmet needs and new findings. The event, which took place in Boston, devoted particular attention to overcoming specificity, efficiency, and delivery challenges associated with the CRISPR/Cas9 system.

Many of these challenges relate to the mechanisms a cell may use to repair CRISPR/Cas-induced double-strand breaks (DSBs). A cell has two pathway choices. Non-homologous end joining (NHEJ), an error-prone ligation process, can result in small insertions and deletions (indels) at cleavage sites, whereas homology-directed repair (HDR) employs homologous DNA sequences as templates to make specific changes for precise repair. In most cells, NHEJ performs the majority of repair events.

Identifying and minimizing off-target events are major challenges. To meet these challenges, the Alt laboratory at Boston Children’s Hospital developed high-throughput genome translocation sequencing (HTGTS), an enzyme- and target-agnostic technique to rapidly expose potential off-target problems. Frederick W. Alt, Ph.D., and colleagues recently described the technique in an article that appeared in Nature Biotechnology.

“The method robustly detects DNA DSBs generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs,” the article read. “HTGTS with different Cas9:sgRNA or TALEN nucleases revealed off-target hotspot numbers for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases more than 10-fold.”

Research scientists and tool suppliers in the life sciences continue to devote resources to CRISPR, which is still a relatively new tool. Much is still unknown, and the community needs a deeper understanding of the technology to better harness CRISPR for discovery and development as well as eventual clinical applications. [Thermo Fisher Scientific]

When HTGTS was used to compare Cas9 nuclease and Cas9 paired nickases, paired nickases showed reduced off-target activity. Paired nickases were also assessed by scientists at Sigma-Aldrich.

“We compared paired nickases to Cas9-FokI nucleases. Paired nickases have about a 10-fold increase in design density, the number of nucleases that target a specific sequence in the selected area,” commented Gregory Davis, R&D manager, molecular biotechnology. “The more nuclease options, the better the chances of finding an active one near site-restricted locations such as disease single-nucleotide polymorphisms (SNPs).”

Like other companies, Sigma-Aldrich is evaluating methods to boost homologous recombination (HR) rates and inhibit NHEJ. Small molecules are being investigated, along with components of the DNA repair machinery such as mRNAs for RAD proteins. Enhancement techniques offer some improvement, but those improvements are not universally applicable to all cell types.

The company recently introduced a nuclease-based kinase knockout lentiviral library, but the challenge is increasing library screening effectiveness for cancer cell lines, which typically demonstrate some level of polyploidy. When the Cas9 nuclease library on the A459 lung cancer cell line was evaluated, a target diploid gene responded with a robust knockout, yet an expected knockout response for another gene was not seen. That particular gene turned out to be tetraploid.

Epigenetically based activators and inhibitors may be another approach, and the company is considering CRISPR-based gene regulation for inhibition or activation, CRISPRi or CRISPRa. Gene regulation may better simulate drugs that suppress activity and prove more effective than the nuclease-knockout method in lentiviral screening applications.

Sigma-Aldrich research scientists Greg Davis, Ph.D., and Fuqiang Chen, Ph.D., discuss genome-editing technologies and best practices.

Measurement Systems

HDR and NHEJ editing events generally occur at low frequencies, necessitating ultrasensitive techniques for detection and quantification of edited alleles. While some studies have relied on NGS, a next-generation PCR technology called droplet digital PCR (ddPCR) is providing researchers with rapid, low-cost, ultrasensitive quantification of both NHEJ and HDR editing events.

ddPCR has already been widely used for high-sensitivity and high-precision applications such as rare cancer mutation detection and copy number analysis, noted Jennifer Berman, Ph.D., staff scientist, Digital Biology Center, Bio-Rad Laboratories.

Since HDR and NHEJ editing events can occur at very low frequency (

“ddPCR is one of the first sophisticated measurement systems for genome editing. The other option is sequencing, which is time-consuming, expensive and out of reach for most people,” explained Bruce Conklin, M.D., a senior investigator at the Gladstone Institute of Cardiovascular Disease and a professor of medicine at the University of California, San Francisco.

The Conklin laboratory works with iPS cells and is primarily interested in HDR, which is typically less than 1% of total alleles. A recent Nature Methods article by the group was the first demonstration that the genome could be changed one base at a time without any mark of an antibiotic reselection marker, a scarless replacement. Populations of cells that have a very rare cell with a single-base change are isolated using ddPCR as a measurement tool, then enriched sequentially, until a pure clone results, in a method termed sib-selection.

“With our method, you can see if the mutation you want is there from the start,” asserted Dr. Conklin. “Single base changes cause many human genetic diseases. To figure out the problem, you want to be able to change one thing and see what happens.”

“We are also looking at ddPCR to quantify HDR and NHEJ simultaneously to isolate conditions where there is more HDR than NHEJ,” he added. “Conditions are different in every cell type, for each location, and we do not understand the rules.”

Droplet digital PCR (ddPCR), a next-generation polymerase chain reaction technology from Bio-Rad Laboratories, can provide rapid, low-cost, ultra-sensitive quantification of both NHEJ- and HDR-editing events.

Application to Animal Models

Mouse models have the potential to quickly screen and build a causal relationship between sequence variations in humans and their phenotypes. Historically, either pronuclear injection of a transgene into a mouse embryo or conventional gene targeting using embryonic stem (ES) cells produced new models.

In 2013, a study led by Rudolph Jaenisch, M.D., a professor of biology at MIT and a founding member of the Whitehead Institute for Biomedical Research, culminated in a published work that was the first to describe a CRISPR/Cas-engineered animal species. CRISPR’s ability to engineer targeted mutagenesis in the genome directly on the zygotes circumvents the need for germline-competent ES cells, and appears to result in more predictable models in a fraction of the previous time and cost.

The simplest way to create a knockout model is to inject CRISPR/Cas reagents, including Cas9 mRNA and a single gRNA, into the mouse embryo. If a knockin alteration is small, the intended mutation can be accommodated into a donor oligonucleotide of the maximal size of 200 bps; for larger alterations that cannot be engineered into a donor oligonucleotide, such as incorporation of a reporter gene or a human sequence, a donor plasmid is often used.

“We are now exploring the use of CRISPR for larger-scale genetic manipulation and humanization of the mouse genome. We do not know yet the size limitation of the genetic manipulation that you can introduce with the CRISPR/Cas technology,” discussed Wenning Qin, Ph.D., associate director of genetic engineering technologies, The Jackson Laboratory.

The Jackson Laboratory uses insertion of a fluorescent reporter gene into the Nanog locus, a gene expressed in early embryos, as the platform for parameter optimization. To determine if there were any off-target effects in addition to the on-target insertion of the reporter gene into the Nanog locus, two HDR mice carrying the reporter gene were genome sequenced, and evaluated minimally for the top 5,000 sites. No off-target effects were observed indicating that the CRISPR/Cas reagent used had a clean off-target profile among the examined sites.

Various means of enhancing the on-target efficiency of CRISPR/Cas9 modifications are being investigated by Taconic Biosciences.  For example, the company is investigating the implementation of Cas9-orthologs to gain more flexibility in the choice of target sequences. It is also evaluating alternative delivery methods as well as experimental design and execution optimization.

To date, analyses have focused on evaluating on-target modifications. Nonetheless, various approaches to enhance specificity—for example, the use of Cas9 nickase, truncated sgRNAs, and Cas9 protein instead of mRNA—are being assessed. According to Jochen Welcker, Ph.D., senior manager of scientific development, improving the generation of more challenging types of alleles is a major development objective, and efficiency issues for specific applications need to be resolved.

For the generation of conditional knockout alleles, one obstacle is the efficient and correct integration of loxP-sites. Using ssODNs (single-stranded oligodeoxynucleotides) as donor material, for example, frequently leads to incomplete integration of the loxP-sites encoded by these ssODNs. The high efficiency of NHEJ-mediated deletion of the genomic region between the two loxP-sites is also a major hurdle as it leads to knockout alleles in up to 30% of the injected zygotes. The generation of complex knockin alleles is limited by the frequency of insertion/replacement of longer sequences by HDR.

Taconic has generated more than 200 humanized models by HDR. A drawback of this approach is the fact that only a single allelic variant can be modeled, while ever increasing amounts of human gene variants are being identified by genome-wide association studies (GWAS). Such variants can now be introduced quickly and efficiently, directly on existing humanized backgrounds by the use of CRISPR/Cas9 genome editing, making it possible to generate a whole range of human allelic variants from a single humanized-mouse model.

The Jackson Laboratory advises that the simplest way to create a knockout mouse model is to inject CRISPR/Cas reagents, including Cas9 mRNA and a single gRNA, into a mouse embryo.

Bioinformatics Needs

“Bioinformatics plays an important role in CRISPR advancement. No-charge informatics packages are available, such as eCRISP, MIT tool, Doench activity scoring, and Zifit, that tackle a portion of the informatics aspects to CRISPR design, and many commercial companies have simple design tools built into their reagent-ordering systems,” stated Eric Rhodes, chief technology officer, Horizon Discovery. “Our new tool, gUIDEbook, a collaboration between Horizon and DesktopGenetics, is the first free application to combine all three aspects of informed design.”

First, all available protospacer adjacent motif (PAM) sites in a given region, which represent a potential gRNA design, must be found. Virtually all CRISPR design tools enable this and differ primarily in how the sequence is entered and user interface complexity. All programs essentially return the same information.

Second, off-target cutting potential by any given gRNA must be determined. Historically, searches found closely related sequences and scores were generated using weighting based on the number of mismatches, their location in the gRNA, and the number of occurrences in the genome. A new finding has demonstrated that “bulges” can occur in the matching of a gRNA to a potential target. More intensive searching is now required to identify all putative binding sites. The unknown is how likely an off-target identified by either method is actually going to be engaged.

The Doench algorithm, which is still early in its development, focuses solely on how much cutting activity a given gRNA design is likely to have. The algorithm calculates predictive scores on the basis of known guides and cutting activities. It does not account for off-target potential, so it has somewhat limited standalone value.


Thermo Fisher Scientific supplies a complete workflow for gene editing and cell engineering that focuses on design, delivery, and analysis. Transfection-grade Cas9 protein and mRNA have been functionally tested in several cell lines, including iPS and ES cells, and both contain a nuclear localization signal (NLS) to aid in delivery. The GeneArt Cas9 Nuclease is extensively purified and quality controlled to remove nonspecific endonucleases and endotoxins.

According to Jason Potter, senior scientist of protein engineering, cell lines vary in how easily they can be transfected. With plasmids and mRNA, the cell must still process the transcripts and make Cas9 complexes before it can act. To simplify the process, the gRNA can be made and complexed with the transfection-grade Cas9 protein in vitro. After it is delivered by lipids or electroporation is used, the Cas9 complex is able to act once it reaches the nucleus. Analysis of the edited cells can then be done using the GeneArt Genomic Cleavage Detection kit or by sequencing.

Lipids, including Lipofectamine 3000, Lipofectamine RNAiMAX, and Lipofectamine MessengerMAX, have been used for delivery of plasmids and RNAs for years. Drawing on this knowledge of Cas9, the company has optimized lipid dosages and protocols for high transfection efficiency and low toxicity. Due to the exposed guide RNA component of the Cas9 complex, RNAiMAX also works for delivery of Cas9 protein. For electroporation, the key consideration is optimizing the voltage and pulse conditions for the cell line.

Gene Editing with Cas9 and Optimal Promoter

The Cas9 (CRISPR associated protein 9) system has gained significant interest due to its relative simplicity and ease of use compared to other genome-engineering technologies, according to many scientists. The CRISPR/Cas9 system requires a complex of the Cas9 protein with a trans-activating RNA (tracrRNA) and a gene-targeting CRISPR RNA (crRNA) or a single guide RNA (sgRNA, a chimeric form of tracrRNA with a crRNA).

Researchers at Dharmacon, now part of GE Healthcare, recently carried out a study on the efficiency of using synthetic crRNA and tracrRNA to introduce gene-editing events when co-transfected with a plasmid expressing Cas9. They explored the use of antibiotic and FACS methods for enrichment of cells that have undergone gene editing, and the use of multiple promoters to increase efficiency of gene editing with Cas9 and synthetic tracrRNA and crRNA.

The researchers reported that utilizing a highly active promoter for Cas9 expression enables better editing in specific cell lines. Enrichment of transiently transfected cells either by fluorescence-activated cell soring or puromycin selection can further improve the yield of edited cells, they added.

In addition, they concluded that efficient gene editing can be achieved with a three-component system: plasmid Cas9 and synthetic tracrRNA and crRNA. They also pointed out that use of synthetic tracrRNA and crRNA is a simplified method for gene editing of one or more genes without requiring any cloning steps, and that the three-component CRISPR/Cas9 system is amenable to high-throughput genome editing applications.

Previous articleAddicts Relapse Because Cocaine Changes the Brain
Next articleA CRISPR Look at Genome Editing