Genetic Engineering & Biotechnology News

AUG 2018

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Page 14 of 33 | Genetic Engineering & Biotechnology News | AUGUST 2018 | 13 be created that link up to 2000 nucleotides and enable high HDR rates, even when short homology arms are used. An additional ad- vantage of ssODNs is that they can be used after standard desalting purification and do not require polyacrylamide gel electropho- resis purification to improve HDR efficiency (Figure 1). Designing an ssODN Template The design of the homology arms is a key factor in stimulating the use of the HDR pathway over NHEJ. To determine how arm length affects template incorporation, we pro- duced a series of templates with arm lengths varying between 27 and 92 nucleotides (Fig- ures 2A & 2B). We also looked at total tem- plate incorporation and HDR-mediated incor- poration. When creating small insertions using Ultramer ® Oligonucleotides (a total maximum donor length of 200 bases), we found good, consistent incorporation at arm lengths be- tween 30 and 60 nucleotides When an ssODN template is used, it is possible to design the template to bind to either the targeting strand or the nontargeting strand. The nontargeting strand is the strand that contains the protospacer adjacent motif (PAM), which is typically a sequence of 2–6 base pairs immediately following the DNA sequence targeted by the Cas9 nuclease. Al- though outcomes vary for different PAM sites, our research shows that HDR rates are often higher when the homology arms are identical to the nontargeting strand (Figure 2C). Selecting a CRISPR Guide RNA The first step in creating the perfect guide for insertion is selecting a suitable PAM site. We found that different PAM sites can have large variations in HDR rate (Figure 3). It is therefore recommended to measure HDR ef- ficiency at a few different PAM sites, since the site closest to the insertion region is not necessarily the best for HDR. However, the insertion cannot be too far away from the cut site. To test the relation- ship between the double-stranded break site and the location of the insertion, we inserted fragments at locations between 0 and 27 base pairs away from the cut site and plotted dis- tance against the rate of HDR (Figure 3). The results show a sharp decrease in HDR rate as the distance from the cut site increases. This means that a balance between the efficiency of the PAM site and its proximity to the inser- tion will need to be determined empirically. When studying HDR rates, it is also im- portant to be aware of the effect of cell type on HDR. We found that the immortal cell line HEK-293 is a suitable cell type for HDR-medi- ated insertion. Conversely, several transformed cells and cancer cells show much lower rates of HDR. Primary cells can perform well in HDR studies, but results are likely to be variable. Error-Free Editing Inserting exogenous sequences into the DNA of living cells is a key strength of CRISPR-Cas9. When reliable insertions with low error rates are required, HDR offers clear benefits over blunt-end incorporation by NHEJ. The results of our HDR optimization studies offers good insight into the parameters that determine how a template is inserted. When it comes to optimization of the CRISPR guide RNA, finding the right PAM site is critical. Both the efficiency of the PAM itself and its proximity to the insertion site determine the rate of HDR incorpora- tion—although some preliminary testing will be required. For the DNA template, Ultramer ssODNs are well suited for use in HDR experiments, as they offer higher efficiency, avoid blunt-end incorporation, and minimize cytotoxicity. Homology arms should be between 30 and 60 nucleotides long and may work slightly better when they match the nontargeting strand. These recommendations can help to improve CRISPR knock-in studies by enabling reli- able, error-free editing. Brian Wang, Ph.D. (, is a genomic tools market development manager at Integrated DNA Technologies. Website: Drug Discovery BRING A WIDE RANGE OF NUCLEIC ACIDS INTO FOCUS. Get high quality downstream QC analysis for your genomics workflow with the Fragment Analyzer ™ from Advanced Analytical - A part of Agilent. Its extraordinary sizing accuracy and wide dynamic range can quickly verify the concentration and size of your DNA and RNA samples— so you can focus on discovery. Find out how QC with the Fragment Analyzer can focus your research. Fragment Analyzer ™ Automated CE System Figure 3. The distance between the double-stranded break and the location of the PAM site is a key parameter for HDR efficiency. Although HDR efficiency tends to decrease as this distance increases, the PAM site nearest the insertion region does not necessarily deliver the highest rate of HDR.

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