Could CRISPR be the Magic Bullet?CRISPR, crispr genome editing system | CRISPR, crispr genome editing system

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

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]

The research community’s rapid acceptance of theCRISPR/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 EditingCongress 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.”

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

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.

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 (<1%), especially HDR in primary or induced pluripotent stem (iPS) cells, ddPCR appears to be a fit for researchers wanting a rapid, sensitive, quantitative readout of editing in cells and tissues. The technique also enables empirical validation of guide RNA efficiency and measurement of the ratio of HDR:NHEJ at a targeted locus.

“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.”

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