Using NGS to Ensure Gene-Editing Success

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Andrea Corona: Hello, everyone, and welcome to Biocompare’s Tech Insights Podcast where we speak to scientific experts about new tools and technology that can help advance your research. I'm Andrea Corona, Associate Editor at Biocompare. And joining us today we have Jason Liu, Field Application Scientist at Roche. He will be talking to us about using next-generation sequencing to ensure gene editing success. Hi, Jason. Thank you for being here today.

Jason Liu: Hello, thank you for having me.

Andrea Corona: Of course, the pleasure is ours. So let's get right into it. My first question for you is, can you tell me what does it mean for a CRISPR edit to be successful? Like, what tools are available to verify that an edit was successful?

Jason Liu: So to me, a successful CRISPR editing means the target gene can be targeted and edited as you wish. At the same time, to minimize any off-target effects, some of the tools that people use to help us to verify the success of CRISPR editing are the T7 endonuclease assays. The other one is called High Resolution Melt Analysis.

So both these measures (PH) actually take advantage of the heteroduplex formations of PCR amplified genes, therefore the editing is a successful. And the other measure is called the TIDE, which is like tracking of indels by decomposition, which is basically you do Sanger sequencing followed by computational analysis. And of course, you know, with the ones for NGS the costs are coming down quite a bit. So the NGS has become the gold standard and popular measure because of its resolution and efficiency.

Andrea Corona: Got it thank you for that. Do researchers have to verify both that the edit was accurate and the CRISPR operated nowhere else in the genome?

Jason Liu: Again, this is a really depends on the research goal of a particular scientist or a lab, right? So if you just want to knock out of a gene to study the function of this gene you know, cultured cell, it may not be too big a concern. It may not be necessary to identify out the off-target cleavage site. However, if you are doing clinical applications, you are developing germline therapies to identify how the off-target cleavage site and understand in their impact on the low model systems or organism it is extremely important because any change you made will pass on to the offspring. So yeah, it really depends on your research goal, what you try to accomplish.

Andrea Corona: Thank you for that. As you said, there are various ways to use NGS to verify that an edit was successful with no off-target cutting, and you advocate for CIRCLE-seq. Can you briefly describe how it works and its advantages over other sequencing-based methods?

Jason Liu: Sure. One of the challenges for off-target identification is that the off-target cleavage could happen anywhere. It's kind of unpredictable, right? So you need a technique which can identify, you know, all potential off-target cleavage sites. Also, the other challenge is that the signal from some of the off-target cleavage is very weak. So you also want this technique to be very sensitive. So CIRCLE-seq, kind of meets both requirements. So it's a whole genome identification of off-target size. At the same time, it’s a very sensitive.

So it was able to achieve this characteristics by, like, a force. You fragmented the genomic DNA into a small fragments in the size about 300 base pairs. So then, you circulize those DNA fragments. So basically, now, you transform the DNA fragments into circles, right? So some of those DNA circles have the Cas9 cleavage site, but most of them don't, right?

So now you treat those DNA as circular molecules with Cas9 reageants, CRISPR reageants. So the one content CRISPR cleavage site can be linearized. They say you kind of linearize those circles. And those linearized molecules has two ends, which containing sequences from both sides of Cas9 cleavage site. So those linearized molecules can be used to build NGS libraries and then you can sequence them.

So because it's the initial assay, so it's simpler, it's also very sensitive. Some of the other alternative techniques for off-target detection, one of them called a Digenome-seq, which is a kind of a brute force sequencing base where you--it’s a in-vitro assay, you know, it’s has a simple workflow. But because the Cas site are not enriched, you have to sequence very deep in order to achieve the sensitivities you need.

The other technique, popular techniques called GUIDE-seq, which is a, well, which is a sensitive, but it's a cell-based assay, which basically require you manipulate, you know, culture, the cells, so it's more involved. So that's why we, like at Roche, we try to advocate for this CIRCLE-seq techniques for off-target identification.

Andrea Corona: Got it. And my last question for you is you recommend the KAPA DNA Library Prep Kit for CIRCLE-seq. What do you look for in a library prep kit to assure that it's the best one for the job?

Jason Liu: Yeah, that's a very good question. The reason, remember, we are trying to identify off-target cleavage site, right? So basically, we are kind of try to identify mutations, right, which means you don't want to identify mutations that are not the true mutations, right?

As we know whenever you do PCR amplification, while PCR is an integral part of any NGS library preparation and techniques, right. So whenever you do PCR, you have the potential to introduce errors. So the benefit of using a high fidelity polymerase is you minimize the chance of introducing errors into your libraries, which could be falsely identified as the off-target cleavage site. That's why with the KAPA HiFi, the Roche Kappa HiFi is the best enzyme, that has the best fidelities on the market, and perfectly fitted for CIRCLE-seq applications.

Andrea Corona: Thank you for all that information and for your time, Jason.

Jason Liu: Oh, you're welcome.

Andrea Corona: If you'd like to know more, please read our related article titled NGS and CRISPR Gene Editing and thank you so much for listening. For more information on products, technologies, and the latest scientific advancements, please visit biocompare.com, and have a great day.

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