Genetic Engineering & Biotechnology News

DEC 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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Page 50 of 69 | DECEMBER 2017 | 17 ments should be analyzed to generate estimates of targeting efficiency. These steps, which come from an application note prepared by scien- tists based at PerkinElmer and other labs, culminate in "visual inspection through digitally represented electro- pherograms and tabular quantitative data," which is enabled by the Lab- Chip Gel Xpress (GX) Touch, a PerkinElmer instrument, notes a PerkinElmer official. "By collaborating with another company, we produced a workflow that allows automation to speed up detection of changes of interest in the DNA of cells which have undergone CRISPR/Cas9 manipulation," states Zhiyeng Peng, Ph.D., application sci- entist, PerkinElmer. "Our new instru- ment completes the analysis of one sample in about one minute. Tradi- tional microcapillary techniques take much longer." "Once the genomic DNA is extracted from the sample of inter- est, the region of interest in DNA is PCR-amplified and then allowed to slowly reanneal to permit formation of heteroduplex strands of DNA. The amplified DNA is then digested with the T7 endonuclease I. The cleavage only takes place at the heteroduplexes, where there are two or more mis- matched base pairs. "The longer fragments of the PCR amplification are the intact DNAs that have not been cleaved by the highly selective T7 DNAse," continues Dr. Peng. "By analyzing the results, the quality and quantity of the CRISPR/ Cas9 reaction can be evaluated." Incorporation of Selection Markers The challenge of ensuring that both copies of the DNA in a diploid cell line have the mutations of interest can be addressed through the use of selection markers. "We have done the hard work of supplying genome-wide knockout kits," details Xuan Liu, Ph.D., head of marketing, OriGene. "We have a kit for every single gene in the human and mouse genomes. The donor vectors, designed to supply do- nor DNA for the CRISPR experiment, also contain selection markers." If a selection marker is used, such as an antibiotic-resistance gene, cells that likely carry a CRISPR/Cas9- induced modification can be isolated. For example, a puromycin resistance gene can be incorporated into a donor vector. Then, after transfection, cells can be challenged with puromycin. The cells that survive are more likely to have been modified as intended. A donor vector can also be engi- neered to contain promoter-less green fluorescent protein (GFP) markers. Such markers can be used to evaluate the promoter strength of the replaced gene in the cells for an event that in- corporates the marker. "One potential workflow involves loxP sites, which can be used to flox out both the GFP and puromycin markers while leaving behind the mu- tated DNA," suggests Dr. Liu. "Ad- vantageously, the same materials can then be used to do another CRISPR experiment to interrupt the gene of interest on the other allele. Thus, an- other allele on the second copy of the DNA can be disrupted with the same materials as the first copy. "Additionally, the GFP can pro- vide a visual marker for cells that have incorporated the DNA of the plasmid vector into its genome. The GFP is an added benefit. It does not have its own promoter; therefore, the GFP is not expressed unless it inserts into a region where a different pro- moter can be used to drive the expression of the GFP." According to Dr. Liu another ben- efit of OriGene's kit is the addition of predesigned genome-wide coverage. "OriGene has done all the hard work of designing both the guide RNAs and corresponding donor vec- tor, assembling them into a kit with a simple protocol. The researchers do not have to become experts on CRISPR to use it in their research," says Dr. Liu. "Following our step- by-step instruction, a person with zero knowledge of CRISPR can utilize this system or kit." B E S T O F C R I S P R 2 017

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