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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16 | DECEMBER 2017 | GENengnews.com has created for library generation of CRISPR oligos provide a complete and general workflow, generating customized plasmid libraries used in high-throughput functional genomics screens, according to Dr. Borgo. End users can now employ a DNA library of their own (or Agilent's) design, run a simple assembly reaction, and move to a plasmid library in less than two hours of hands-on work, he points out. Because there are a number of ex- isting solutions, users can introduce errors and biases into the library dur- ing characterization, which leads to additional cost and uncertain results when the actual screening is performed, maintains Dr. Borgo. "The SureGuide librar- ies developed by Agilent eliminate this quality loss, as well as the associated uncertainty," he says. "In general, our solution dras- tically simplifies the most general steps in a pooled CRISPR library workflow and does so in a way that not only maintains the quality of the library, but improves it from step to step." Measuring Targeting Efficiency Other technologies aim to facilitate and accelerate processes that evaluate whether cell lines incorporate CRIS- PR-induced mutations. Normally, after a CRISPR experiment, scientists must isolate, grow, and analyze several subclones from cell lines to deter- mine whether genetic modifications that had been intended were actually implemented. "The technology of our process is a basic T7 endonuclease I assay," says Kyle Luttgeharm, Ph.D., application scientist, Advanced Analytical Tech- nologies (AATI). "AATI has carefully optimized this assay to ensure that the cleavages produced are actually match- ing the probability of what should be happening in that test tube. We recently published a paper on all of our work in the journal BioTechniques." The T7 endonuclease I recognizes mismatches in DNA heteroduplexes by detecting the "bubble" that forms in the double-stranded DNA. "AATI has developed a kit using a highly active T7 endonuclease, AccuCleaveâ„¢ T7CE," notes Dr. Luttgeharm. "In combination with our instrument, the Fragment An- alyzer Automated CE System, and an- other kit (CRISPR Discovery Gel Kit), the AccuCleave endonuclease facilitates a high-throughput ability to rapidly screen for clones likely to contain the desired mutations." In a traditional research setting, the steps to determine the mutational status of clonal lines after the CRISPR experiment would be to extract genom- ic DNA, PCR amplify the fragment of interest, and then sequence these frag- ments. Following these steps for many clones is a laborious, protracted, and expensive proposition. Using the meth- ods and instrumentation from AATI can expedite the process, reducing the amount of resources employed to find the correctly modified cell line. "Some of our customers in the phar- maceutical industry use this process to screen for modified cell lines," informs Dr. Luttegeharm. "AATI happily works with our customers to design a work- flow that minimizes expenses of these processes. We also work with compa- nies that develop CRISPR applications themselves, to assist them in evaluating the efficacy of their products." The Use of T7 Endonuclease to Expedite Screening The machinery of CRISPR/Cas9 was discovered in bacteria and archaea, microorganisms in which CRISPR/Cas9 serves as an adaptive immune sys- tem. This system, which pro- tects against foreign genetic material, has the previously mentioned component, T7 endonuclease I, which recog- nizes and cuts double-strand- ed DNA where there are two or more mismatched DNA base pairs. Efforts to engineer deletions or insertions in a DNA site by CRISPR/Cas9 experiments often result in two or more mismatched base pairs. The cutting action of T7 endo- nuclease I can be exploited experi- mentally, for example, to assess the targeting efficiency of CRISPR/Cas9. Cells that have been subjected to ge- nome editing are lysed to permit the extraction of genomic DNA, which is amplified (for the region of interest) with PCR. After PCR amplification, the DNA undergoes denaturing and reannealing, yielding various reanneal- ing products, some of which constitute heteroduplexes, which are digested with T7 endonuclease I. Then, the un- digested DNA and the digestion frag- B E S T O F C R I S P R 2 017 Genome Engineering: CRISPR Proving More User-Friendly Continued from page 14 The challenge of ensuring that both copies of the DNA in a diploid cell line have the mutations of inter- est can be addressed through the use of selection markers.

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