Genetic Engineering & Biotechnology News

AUG 2018

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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12 | AUGUST 2018 | Genetic Engineering & Biotechnology News | Brian Wang, Ph.D. In recent years, CRISPR-Cas9 genome edit- ing has enjoyed spectacular success because it combines flexibility and usability. The Cas9 nuclease need only be loaded with a guide RNA to direct it to almost any genomic loca- tion, where it will create a double-stranded break that may serve as an insertion point for exogenous DNA. After a cell's genomic DNA is cut, the cell's DNA repair mechanisms will repair the break, splicing together the loose ends, whether donor DNA is present or not. If donor DNA is present, the repair may in- corporate the exogenous sequence between the (formerly) loose ends. To carry out the repair process, eukary- otic cells have two mechanisms at their dis- posal: • Nonhomologous end joining (NHEJ) can repair a double-stranded break without requiring homologous sequences on both sides of the break. This makes NHEJ a more straightforward form of repair, but it also in- creases the risk of errors, such as insertions or deletions, at the cut site. • Homology-directed repair (HDR), or what has been called "true genome editing," relies on the presence of a template with DNA "arms" homologous to each end of the break. These sequences are used to guide repair and can fix breaks with a vastly reduced error rate. Due to its greater reliability in DNA repair, HDR is the preferred mechanism if CRISPR- Cas9 is used to insert (or knock-in) exogenous DNA sequences, such as selectable markers or fluorescent tags. Therefore, improving the rate of HDR is a key challenge in achieving reliable insertions. In this tutorial, we describe a series of strategies for obtaining higher rates of HDR based on experimental data from our detailed CRISPR optimization studies. Single- or Double-Stranded DNA? Although most researchers use double- stranded DNA, both single- and double- stranded DNA templates are suitable for CRISPR-mediated insertion. However, using a double-stranded template is more likely to result in blunt-end incorporation by NHEJ with duplication of the homology arms. To reduce the risk of blunt-end incor- poration, and thus achieve a higher rate of template incorporation by HDR, single- stranded oligo deoxynucleotides (ssODNs) can be used. Single-stranded templates can Improving CRISPR-Cas9's Rate of Homology-Directed Repair Integrated DNA Technologies Suggests Strategies to Boost Efficiency, Avoid Blunt-End Incorporation, and Reduce Toxicity Drug Discovery Tutorial Figure 1. Amplicon sequencing shows high rates of insertion of Ultramer ssODN template (top), with up to 50% of insertions being repaired by HDR (bottom). PAGE purification does not appear to improve insertion compared to standard desalting purification. Figure 2. (A) Design of DNA templates with homology arms between 27 and 97 nt. (B) Effect of the arm length of different templates on HDR rate. HDR rate (bars) was determined by using an EcoRI restriction site as the inserted sequence and measuring the percentage of EcoRI cleavage. Total editing (dots) was determined by T7EI digestion. (C) The effect of strand homology of the template and the location of the cut site on HDR rate. RNP: ribonucleoprotein. B C A

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