In today's post, we have the pleasure of presenting an exclusive interview with our very own Stephanie Scherer PhD, Manager of Scientific Engagement at BioSkryb Genomics. She is our in-house CRISPR expert and enthusiast, and she will share her insights on why CRISPR is such an important scientific advancement.
Throughout this interview, Stephanie provides an in-depth look at the evolution of gene editing offering valuable perspectives and practical insight. Whether you’re interested in the gene editing field, looking to gain some expert knowledge, or simply curious about the innovation in developing off-target assays since the introduction of CRISPR/Cas9, this Q&A session is sure to be informative.
Without further ado, let's dive into our conversation with Stephanie.
Why is CRISPR important?
Q: It seems that over the past decade or so, CRISPR has become a ubiquitous term. From your perspective, why is CRISPR such an important scientific advancement?
A: To fully answer that question, I’m going to take a step back and talk a little bit about what gene editing is and the history that preceded the wide adoption of CRISPR. Simply put, gene editing is the method used to purposefully create changes in the DNA sequence of a cell or organism’s genome. There is a plethora of applications for gene editing, including things like creating reporter cell lines for drug screening, engineering disease models for preclinical research, or even developing cell therapies for diseases. Scientists have been performing gene editing for many years, even before the discovery of CRISPR/Cas9 as a gene editing tool. Meganucleases, zinc finger nucleases (ZFNs), and transcription activator-like (TAL) effector nucleases (TALENs) are all gene editing technologies that helped set the stage for CRISPR/Cas9. While there are unique properties of each of these gene editing technologies, they are all nuclease proteins that create double-stranded DNA breaks (DSBs), which in turn initiate a cell’s innate DNA-repair pathways and result in gene edits. What makes them all especially useful for gene editing is that scientists can engineer the DNA-binding regions of these nucleases to target specific genomic sequences – thereby allowing gene editing to occur virtually anywhere in the genome. While these gene editors were and continue to be invaluable tools for gene engineering, one drawback they share is that designing and producing bespoke protein nucleases per gene target site is complicated and time consuming. This is why the discovery of CRISPR/Cas9 for gene editing was so groundbreaking. Unlike meganucleases, ZFNs, and TALENs, CRISPR/Cas9 uses a guide RNA molecule to direct the Cas9 nuclease protein to a specified target sequence in the genome. Because RNA molecules are relatively simple to design and manufacture compared to proteins, CRISPR/Cas9 presented a nuclease system that was more feasible to use for each new DNA target and this property contributed strongly to its wide adoption.
Off-targets
Q: Often times when CRISPR gene editing is discussed, the topic of off-target edits also comes up. Can you speak a little bit about what off-target edits are and how they happen?
A: Absolutely. Off-target edits are DNA changes that occur at genomic locations other than the CRISPR target location. Off-target editing can occur when a guide RNA directs Cas9 to a genomic sequence that has slightly less than perfect sequence homology with the guide RNA. However, there have also been reports of off-target editing at sites that have no apparent sequence homology to the guide RNA that was used. Experimental factors can also influence the prevalence of off-target edits, for example, impurities in the guide RNA preparation used or previously unrecognized sequence differences between the genomes of the edited cells and the reference genome used during guide RNA design. It ’is really important to understand what, if any, off-target modifications occurred in CRISPR/Cas9-edited cells because they can negatively impact interpretation of experimental results when using these cells in research. In the context of gene-edited medicines, off-target edits can reduce efficacy or pose serious safety risks.
Measuring off-targets
Q: Because, as you said, understanding off-targets is so important when performing gene editing, how do scientists measure off-target edits?
A: That is a great question. Developing assays to accurately predict and detect CRISPR off-target edits has been a huge focus of the gene editing community – I think at this point there are over 20 different methods for off-target analysis! The methods can broadly be categorized in three groups – in silico prediction methods, cell-free experimental methods, and cell-dependent experimental methods.
In silico methods
In silico prediction methods, such as Cas-OFFinder, CROP-IT, and others, predict potential off-target sites based on sequence homology with a guide RNA of choice. These methods are easy to use and are often freely available online, but they are unable to predict off-target locations that are independent of guide RNA sequence homology. They also rely on reference genomes, which may be different than the genome found within the targeted cell.
Cell-free methods
Cell-free off-target methods, such as SITE-Seq, CIRCLE-Seq, and others, combine genomic DNA with CRISPR/Cas9 complexes in vitro and then use various methods to detect where double-stranded DNA breaks occurred. These methods are relatively quick to implement and have been widely adopted, however they do not perfectly mimic the cellular context in which gene editing occurs. For example, you can imagine DNA that is packaged into nucleosomes or bound by transcription factors could be less accessible to CRISPR/Cas9 machinery.
Cell-dependent methods
Cell-dependent methods such as GUIDE-seq, DISCOVER-Seq, and others introduce CRISPR/Cas9 complexes into cells and then most methods use techniques that either directly or indirectly label the locations where double stranded breaks occurred. These methods have the benefit of examining off-targets in the biologically relevant context of the cell, but some methods suffer from inefficient or challenging DSB labeling techniques.
Because no off-target method is perfect at predicting all possible off-target locations, scientists often use multiple assays to examine the off-target editing profile of their chosen gene editor. Also, it is important to recognize that all these assays are designed to predict where in a genome CRISPR/Cas9 may produce an off-target edit; they do not actually measure what those edits end up being in a CRISPR/Cas9-edited cell product. To do that, most groups perform deep sequencing of an amplicon panel comprised of potential off-target locations nominated by the previously discussed assays.
Developments in gene editing
Q: Wow, there has clearly been a ton of innovation in developing off-target assays since the introduction of CRISPR/Cas9. I would like to build off the theme of innovation. What other developments in the gene editing field excite you?
A: Generally, it is a field that is innovating and developing so quickly that it is hard not to be excited about many things. Late last fall we got the first FDA-approval of a CRISPR-based medicine with Casgevy® from Vertex and CRISPR Therapeutics, likely paving the way for more CRISPR-based therapy approvals in the future. This came just 11 years after CRISPR/Cas9 was introduced as a gene editing tool, which just goes to show how rapidly this field moves. There has also been a ton of exciting developments of new gene editors with unique capabilities. Now we have multiple varieties of Cas proteins used in gene editing, such as Cas12 which can target sequences in the genome that are untargetable by Cas9, and Cas13 which targets RNA instead of DNA. Several groups have used protein engineering to generate new ribonucleoprotein complexes based on CRISPR/Cas9 but with entirely new functionalities. Some examples include base editors and prime editors, neither of which create DNA double-stranded breaks at all. Base editors allow for precise conversion of one base pair to another and prime editors allow for the introduction or deletion of short sequences without relying on DSBs and donor DNA templates. And, just like how CRISPR was a completely novel addition to an existing list of gene editing tools over a decade ago, there will likely be other instances of novel, non-CRISPR tools being added to the gene editing toolbox in the future. In fact, just this summer, scientists from the Arc Institute in California published their discovery of bridge RNAs as a gene editing tool for programmable DNA insertion, excision, or inversion. I am excited to see how these and other yet undiscovered technologies transform the gene editing field in the coming years and decades.
New assays for off-targets
Q: With all these new editors, do you think there will be a need to develop new assays for off-targets for all of them?
A: There will absolutely be a need to identify off-target events for new editors and groups have already been developing new assays to measure the off-target activity of some of the editors I mentioned. However, considering the rapid development of this field, developing custom assays for every type of editor may soon become impractical. This is one reason why I think BioSkryb’s products and services offer unique solutions for characterizing gene edited cells. BioSkryb specializes in enabling single-cell DNA sequencing with ResolveDNA® products and DNA plus RNA sequencing from the same cell with ResolveOME™ products. Because ResolveDNA offers nearly complete (>95%) coverage of single-cell genomes, it can be a powerful tool for deeply characterizing both on- and off-target gene edits regardless of the type of gene editor used, and we already have some customers using it for this purpose. Historically, whole genome sequencing (WGS) of edited cells was only useful for characterizing off-target edits in clonal populations because of lack of sensitivity for detecting rare off-target edits in heterogenous populations of cells. Because ResolveDNA enables whole genome sequencing at single-cell resolution it can detect off-target edits that otherwise would be below the limit of detection of WGS of cell populations. We have also shown that using ResolveDNA enables detection of off-target insertions and deletions (indels), copy number variation, and translocations in CRISPR/Cas9 treated cells. Additionally, taking a single-cell approach unlocks the ability to make zygosity calls for on- and off-target edits and understand what edits co-exist in cells, which can be especially helpful in programs that edit multiple targets at once. So basically with ResolveDNA scientists can answer the relatively simple question of “how do my edited cells look different than non-edited cells?” across multiple DNA mutation types on a whole genome scale. While I think there are a lot of upsides to this approach, it is still important to recognize that one of the drawbacks is sequencing cost associated with WGS when compared to targeted sequencing. With that said, I have been happy to see the advancements in sequencing technology in recent years with a continual trend towards less costly sequencing, which I expect will make single- cell WGS attainable for more scientists moving forward. I am also excited to see how scientists apply multiomic approaches for an even richer understanding of gene edited cells. For example, ResolveOME offers the possibility to not only characterize what on- and off-target edits occur in a gene edited cell, but to also understand the impacts of those edits on the transcriptome of that same cell. All-in-all I can’t wait to see where the field of gene editing goes next and how BioSkryb can support.