How specific are CRISPR-cas9 cuts?

How specific are CRISPR-cas9 cuts?

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CRISPR-cas9 uses a string of RNA that matches with DNA and makes a double stranded cut at that point.

If the RNA is just a few letters in length, the enzyme would cut DNA in many places. It would be unspecific. But if you can make the RNA string very long, it would only cut at the exact place where you want to cut.

So how long can you make the RNA sequences? And is this enough to avoid unintended cuts?

The problem of off-targets in CRISPR/Cas is often discussed. It was shown that the system allows mismatches up to five basepairs. For your question, if it is helpful to elongate the gRNA: it was shown that truncating the RNA enhances the specificity more than elongating (see also here).

So what can we do? Well, there are different methods to improve the specificity. The simplest may be the usage of lower dosages of the CRISPR/Cas system. Another method is to use altered PAM motifs.

A more complicated method is to convert the Cas9 into a nickase. In this strategy you would use two different gRNAs for two different target sites and introduce ssbreaks. A modification of this method is to completely knock-out the enzymatic activity of Cas9 and fuse it to a FokI nuclease which will cut the DNA only when it dimerizes.

There are several advantages in these methods, however, you may also loose on-target effects.

There is another method which, in my opinion, is really awesome. It is possible to alter the energetics of the DNA binding site resulting in high-fidelity variants of Cas9 (HF1 and HF2)

So, I hope I could help you. There are other and even more effective ways to increase CRISPR/Cas specificity instead of altering the gRNA. If you're interested, have a look on the references.

Evaluation of CRISPR/Cas9 site-specific function and validation of sgRNA sequence by a Cas9/sgRNA-assisted reverse PCR technique

The effective application of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system in biology, medicine and other fields is hindered by the off-target effects and loci-affinity of Cas9-sgRNA, especially at a genome-wide scale. In order to eliminate the occurrence of off-target effects and evaluate loci-affinity by CRISPR/Cas9 site-specific detection and screening of high-affinity sgRNA sequences, respectively, we develop a CRISPR/Cas9-assisted reverse PCR method for site-specific detection and sgRNA sequence validation. The detection method based on PCR can be used directly in the laboratory with PCR reaction conditions, without the need for an additional detection system, and the whole process of detection can be completed within 2 h. Therefore, it can be easily popularized with a PCR instrument. Finally, this method is fully verified by detecting multiple forms of site mutations and evaluating the affinity of a variety of sgRNA sequences for the CRISPR/Cas9 system. In sum, it provides an effective new analysis tool for CRISPR/Cas9 genome editing-related research. A CRISPR/Cas9-assisted reverse PCR method was developed for Cas9/sgRNA site-specific detection and sgRNA sequence validation. The technique detects target DNA in three steps: (1) target DNA is specifically cut by a pair of Cas9/sgRNA complexes (2) the cleaved DNA is rapidly linked by T4 DNA ligase (3) the ligated DNA is efficiently amplified by PCR (PCR or qPCR).

Keywords: CRISPR/Cas9 Site-specific detection sgRNA validation.

Conflict of interest statement

The authors declare that they have no conflict of interest.


A CRISPR/Cas9-assisted reverse PCR method was developed…

A CRISPR/Cas9-assisted reverse PCR method was developed for Cas9/sgRNA site-specific detection and sgRNA sequence validation.…

Schematic illustration of CARP detection

Schematic illustration of CARP detection

CARP detection of single-base mutation…

CARP detection of single-base mutation (PCR). a Determining the ability of CARP to…

CARP detection of single-base mutation…

CARP detection of single-base mutation (qPCR) (the error bars represent technical replicates and…

Detecting single-nucleotide specificity of Cas9-sgRNA…

Detecting single-nucleotide specificity of Cas9-sgRNA with qPCR-based CARP. a Detailed location of common…

Evaluating single-nucleotide specificity of sgRNA…

Evaluating single-nucleotide specificity of sgRNA with qPCR-based CARP. a Detailed location of single-base…

Exploring the effects of different…

Exploring the effects of different sgRNAs on cleavage efficiency simultaneously. a qPCR logarithmic…

Function and Mechanism of CRISPR in Prokaryotes

CRISPR provides prokaryotes with acquired immunity against invasive genetic elements. These loci incorporate genetic material from viruses and plasmids, and use it to target foreign genetic elements in a sequence-specific manner. The steps involved in protecting against foreign genetic elements using the CRISPR/Cas system are acquisition of spacer DNA from the invading virus, biogenesis of CRISPR RNA (crRNA), which will allow for recognition of foreign DNA, and interference, in which the invasive DNA is recognized and cleaved.

Acquisition of Spacer DNA

When a prokaryotic cell is invaded by a virus, portions of the viral DNA are sampled and incorporated into the spacer regions of the CRISPR locus. The nuclease enzymes Cas1 and Cas2 are involved in spacer acquisition in E. coli and likely all CRISPR/Cas systems, as they are the only Cas proteins found to be conserved across all systems. The DNA that is sampled and incorporated into CRISPR loci as spacers may be located next to protospacer adjacent motifs (PAMs) within the viral genome PAMs are important in selection of foreign nucleic acids in type I and type II systems. Spacers are usually added next to the leader sequence, though the new spacer may also be inserted randomly into the repeat-spacer array.

CrRNA Biogenesis

During the interference stage, crRNAs associate with Cas proteins to form a complex that recognizes, targets, and destroys viral genetic material. The crRNA basepairs with the complementary sequence in the viral DNA, marking it for destruction. Recognition of the PAM sequence may also be required for recognition of the foreign DNA. In type II systems, Cas9 carries out the interference step using both a crRNA and tracrRNA which allows it to recognize foreign DNA. In addition to recognizing target sequences, Cas9 has endonuclease activity and cleaves the foreign DNA.

The figure depicts how the CRISPR/Cas system defends against foreign genetic elements in prokaryotes.

CRISPR-Cas9 can cut RNA, too

Würzburg scientists discovered that the so-called Cas9 DNA scissors from Campylobacter can also readily target RNA. From the left: Prof. Dr. Cynthia Sharma, Sara Eisenbart, Thorsten Bischler, Belinda Aul from the Institute of Molecular Infection Biology (IMIB) and Prof. Dr. Chase Beisel from the Helmholtz-Institute of RNA-based Infection Research (HIRI) in Würzburg. Credit:Hilde Merkert, IMIB

The ability to edit genes at will, whether to reverse genetic diseases or improve food and energy crops, is undergoing a revolution. It is being driven by CRISPR-Cas9, a technology modeled on a cellular mechanism found in bacteria. CRISPR-Cas9 recognizes and cuts foreign genomic material from invading viruses and thus protects the bacteria from being infected.

The Cas9 protein acts as a pair of scissors, while other parts of the system guide Cas9 to the sections of DNA to be cut. Scientists have harnessed these molecular scissors in combination with artificial guides to specifically modify genes, not only in bacteria but also in plants and animals.

While the Cas9 scissors are known to cut DNA, researchers from the Julius-Maximilians-Universität Würzburg (JMU) and the Helmholtz Institute for RNA-based Infection Research (HIRI) in Germany have demonstrated that the Cas9 protein of the food-borne pathogen Campylobacter jejuni also cuts RNA.

"The protein is also capable of cutting ribonucleic acids—RNA, for short," says Prof. Cynthia Sharma from the JMU Institute for Molecular Infection Biology (IMIB). "Not only that, but we found that we could also program Cas9 to target and cut specific RNA molecules."

RNA plays a central role in all forms of life. A major role of RNAs is to serve as messenger of genomic material in the cell. Genes encoded in the DNA are extracted by transcribing them into RNA. The RNA then serves as template for the translation of this information into proteins. The ability to target RNA instead of DNA expands how Cas9 scissors can be used. Potential uses include controlling which genes are activated or deactivated, and combating human viruses, and rapidly detecting infectious agents.

The researchers discovered this function while looking at molecules that interact with the Cas9 in Campylobacter. These included numerous RNAs from the cell. Further analyses showed that Cas9 not only bound to RNA, but could also cut it as it does with DNA. The researchers determined that it could be easily instructed to cut specific RNAs.

"The finding was surprising, given that Cas9 is thought to naturally target DNA only," says Prof. Chase Beisel, who recently joined HIRI from NC State University (USA) and has been collaborating with Prof. Sharma on the project.

While the researchers made this finding with the Cas9 protein from Campylobacter, two other groups of researchers recently reported similar findings with Cas9s from two other bacteria. This raises the possibility that this fascinating new discovery could be a general trait of Cas9 proteins in nature. Another question raised by this study is whether the ability of Cas9 to target RNA has any physiological roles in Campylobacter. For instance, evidence is accumulating that CRISPR-Cas systems might not only serve to combat infections, but might rather be naturally involved in controlling which genes in Campylobacter are turned on and off. Prof. Sharma and Prof. Beisel agree: "We continue to be amazed by what Cas9 is capable of doing and what new applications and technologies these insights create."

CRISPR discovery paves the way for novel COVID-19 testing method

The novel platform LEOPARD has the potential to detect a variety of disease-related biomarkers in just one test. Credit: Sandy Westermann / HIRI

Most conventional molecular diagnostics usually detect only a single disease-related biomarker. Great examples are the PCR tests currently used to diagnose COVID-19 by detecting a specific sequence from SARS-CoV-2. Such so-called singleplex methods provide reliable results because they are calibrated to a single biomarker. However, determining whether a patient is infected with a new SARS-CoV-2 variant or a completely different pathogen requires probing for many different biomarkers at one time.

Scientists from the Helmholtz Institute for RNA-based Infection Research (HIRI) and the Julius Maximilians University (JMU) in Würzburg have now paved the way for a completely new diagnostic platform with LEOPARD. It is a CRISPR-based method that is highly multiplexable, with the potential to detect a variety of disease-related biomarkers in just one test.

LEOPARD, which stands for "Leveraging Engineered tracrRNAs and On-target DNAs for PArallel RNA Detection," is based on the finding that DNA cutting by Cas9 could be linked to the presence of a specific ribonucleic acid (RNA). This link allows LEOPARD to detect many RNAs at once, opening opportunities for the simultaneous detection of RNAs from viruses and other pathogens in a patient sample.

The study published today in Science was initiated by Chase Beisel, professor at JMU and research group leader at HIRI, and Professor Cynthia Sharma from JMU's Institute of Molecular Infection Biology (IMIB). "With LEOPARD, we succeeded in detecting RNA fragments from nine different viruses,' says Beisel. "We were also able to differentiate SARS-CoV-2 and one of its variants in a patient sample while confirming that each sample was correctly collected from the patient."

CRISPR-Cas9 is principally known as a biomolecular tool for genome editing. Here, CRISPR-Cas9 function as molecular scissors that cut specific DNA sequences. These same scissors are naturally used by bacteria to cut DNA associated with invading viruses. Whether editing genomes or eliminating viruses, Cas9 cutting is directed by guide RNAs. The guide RNAs found in bacteria must pair with a separate RNA called the tracrRNA. The RNA couple then can work with Cas9 to direct DNA cutting.

An unexpected discovery

The tracrRNA was thought to only pair with guide RNAs coming from the antiviral system. However, the Würzburg scientists discovered that the tracrRNA was pairing with other RNAs, turning them into guide RNAs. Cynthia Sharma, Chair of Molecular Infection Biology II at the IMIB and spokesperson of the Research Center for Infection Diseases (ZINF) at JMU was astounded by this discovery: "When we searched for RNAs binding to Cas9 in our model organism Campylobacter, we surprisingly found that we detected not only guide RNAs, but also other RNA fragments in the cell that looked like guide RNAs. The tracrRNA was pairing with these RNAs, resulting in "non-canonical" guide RNAs that could direct DNA cutting by Cas9."

The LEOPARD diagnostic platform builds on this discovery. "We figured out how to reprogram the tracrRNAs to decide which RNAs become guide RNAs," says Beisel. "By monitoring a set of matching DNAs, we can determine which RNAs were present in a sample based on which DNAs get cut. As part of the ongoing pandemic, LEOPARD could allow a doctor to figure out whether the patient is infected with SARS-CoV-2, if it's a unique variant, and whether the sample was correctly taken or needs to be repeated—all in one test."

In the future, LEOPARD's performance could dwarf even multiplexed PCR tests and other methods. "The technology has the potential to revolutionize medical diagnostics not only for infectious diseases and antibiotic resistances, but also for cancer and rare genetic diseases," says Oliver Kurzai, director of JMU's Institute of Hygiene and Microbiology, which provided patient samples for the study.

"The work highlights the excellent collaborative and interdisciplinary research taking place here in Würzburg," says Jörg Vogel, director of IMIB and HIRI, a joint facility of JMU with the Helmholtz Center for Infection Research in Braunschweig. "LEOPARD impressively demonstrates that we can cover the full spectrum of complementary cutting-edge research in Würzburg, from the fundamentals of RNA research to clinical applications."

Simple technology makes CRISPR gene editing cheaper

University of California, Berkeley, researchers have discovered a much cheaper and easier way to target a hot new gene editing tool, CRISPR-Cas9, to cut or label DNA.

Some DNA sequences appear multiple times in the genome. Here, an RNA guide probe labels repetitive regions in the nucleus of a sperm cell from the frog Xenopus laevis.

The CRISPR-Cas9 technique, invented three years ago at UC Berkeley, has taken genomics by storm, with its ability to latch on to a very specific sequence of DNA and cut it, inactivating genes with ease. This has great promise for targeted gene therapy to cure genetic diseases, and for discovering the causes of disease.

The technology can also be tweaked to latch on without cutting, labeling DNA with a fluorescent probe that allows researchers to locate and track a gene among thousands in the nucleus of a living, dividing cell.

The newly developed technique now makes it easier to create the RNA guides that allow CRISPR-Cas9 to target DNA so precisely. In fact, for less than $100 in supplies, anyone can make tens of thousands of such precisely guided probes covering an organism’s entire genome.

The process, which they refer to as CRISPR-EATING – for “Everything Available Turned Into New Guides” – is reported in a paper to appear in the August 10 issue of the journal Developmental Cell.

As proof of principle, the researchers turned the entire genome of the common gut bacterium E. coli into a library of 40,000 RNA guides that covered 88 percent of the bacterial genome. Each RNA guide is a segment of 20 RNA base pairs: the template used by CRISPR-Cas9 as it seeks out complementary DNA to bind and cut.

The researchers created hundreds of RNA guide probes for a small region of the genome of the frog Xenopus laevis, attached them to CRISPR-Cas9 with a fluorescent label, and successfully tagged that region of DNA in a nucleus so that it can be followed in living samples.

These libraries can be employed in traditional CRISPR-Cas9 editing to target any specific DNA sequence in the genome and cut it, which is what researchers do to pin down the function of a gene: knock it out and see what bad things happen in the cell. This can help pinpoint the cause of a disease, for example. The process is called genetic screening and is done in batches: each of the thousands of probes is introduced into a single cell on a plate filled with hundreds of thousands of cells.

“We can make these libraries for a lot less money, which makes genetic screening potentially accessible in organisms less well studied,” such as those that have not yet had their genomes sequenced, said first author Andrew Lane, a UC Berkeley post-doctoral fellow.

Real-time cell monitoring

But Lane and colleague Rebecca Heald, UC Berkeley professor of molecular and cell biology, developed the technology in order to track chromosomes in real-time in living cells, in particular during cell division, a process known as mitosis. This is part of a larger project by Heald to find out what regulates the size of the nucleus and other subcellular components as organisms grow from just a few cells to many cells.

“This technology will allow us to paint a whole chromosome and look at it live and really follow it in the nucleus during the cell cycle or as it goes through developmental transitions, for example in an embryo, to see how it changes in size and structure,” Heald said.

The new technique uses standard PCR (polymerase chain reaction) to generate many short lengths of DNA from whatever segment of DNA a researcher is interested in, up to and including an entire genome. These fragments are then precisely snipped at a region called a PAM, which is critical to CRISPR binding. Simple restriction enzymes are then used to cut each piece 20 base pairs from the PAM end, generating the exact size of RNA guide that CRISPR uses in searching the genome for complementary sites. These guide RNAs are then easily incorporated into the CRISPR-Cas9 complex, yielding tens of thousands of probes for labeling or cutting DNA.

“By using the genome itself as a source for guide RNAs, their approach puts the creation of libraries that target contiguous regions in reach of almost any lab,” said Jacob Corn, managing and scientific director of the Innovative Genomics Initiative at UC Berkeley. “This could be very useful for genome imaging and certain kinds of screens, and I’m very interested to see how it enables biological discovery using Cas9 tools.”

Lane and Heald’s coauthors are Magdalena Strzelecka, Andreas Ettinger, Andrew W. Grenfell and Torsten Wittmann. This work was supported by the National Institutes of Health.


How specific are CRISPR-cas9 cuts? - Biology

The beauty of the system is that it consists of just two elements: a guide RNA, which binds the targeted DNA, and the DNA cutting nuclease Cas9, which complexes with the guide RNA. Both elements can be delivered into cells using a single vector. The highly efficient and cost-effective technique is so versatile it is set to transform a host of disciplines, from medicine to agriculture and industrial biotechnology.

Human health

The use of CRISPR-Cas9 carries enormous possibilities to further advance human health and well-being. While the ultimate aim is to eradicate diseases, the majority of work using the technique is still at the research stage. For example, CRISPR is having a huge impact on how potential drug targets for cancer and other conditions are discovered, as it enables researchers to hone in on and edit specific genes much more efficiently and less expensively than with previous genome editing methods.

In February 2016, research scientists in London were granted permission to use CRISPR-Cas9 to edit healthy human embryos in a quest to develop treatments for infertility. This was the world’s first endorsement of such research and sets a strong precedent for allowing similar applications to go forward.

So who will win the race to develop the first CRISPR therapeutic? In 2015, Editas Medicine announced its plans to use CRISPR to try to treat a rare form of blindness known as Leber congenital amaurosis.

More recently, in June 2016, approval was given by a federal panel in the USA for the first ever clinical trial of CRISPR to create genetically altered immune cells for the treatment of cancer. Scientists at the University of Pennsylvania plan to use CRISPR in the pioneering technology of Chimeric Antigen Receptor (CAR)-T cell therapy to engineer T-cells to make them more effective at identifying and destroying three types of cancerous tumour cells. CAR-T cell therapy has received striking early success against certain blood cancers in patients who have failed to respond to other treatments. The use of CRISPR-Cas9 in combination with CAR T-cells could prove to be a game changer in saving the lives of those with previously untreatable cancers.

Elsewhere, researchers are exploring the possibility of using CRISPR to cure HIV, with some success already being reported both in isolated human cells and in mice and rat models, while another team at Berkeley is attempting to use the technique to correct the mutation that causes sickle cell anaemia.

Crop research

While the spotlight is undoubtedly on CRISPR’s potential role in therapy, it is also being used as a genome editing tool in a diverse range of crops, including wheat, rice, soybeans, potatoes, sorghum, oranges and tomatoes. In a recent study, John Innes Centre scientists used the technology to make targeted edits to two UK crops – a broccoli-like brassica and barley – and these edits are shown to be preserved in subsequent generations. CRISPR has also been used to create grapevines that are resistant to downy mildew disease and wheat that is resistant to powdery mildew.

Industrial biotechnology

There is huge potential for the use of CRISPR in industrial biotechnology. A major player in the field is Caribou Biosciences who are working to improve microbial production strains to generate better cell factories for fermentation. They are also using the technique to unlock the potential of microbes to bioproduce chemicals and enzymes never previously produced by fermentation.

The Swiss synthetic biology company Evolva is also adding CRISPR to its toolkit, which promises a major boost to its search for new ingredients and specialty chemicals with brewing and engineered microorganisms. Evolva’s products range from nootkatone, a flavour ingredient of grapefruit that could help stop the spread of Zika, to agricultural bioactives and components of next-generation materials.


Despite the extraordinary power and potential applications of the technique, there is concern in the scientific community that CRISPR might inadvertently alter regions of the genome other than the intended ones, thereby affecting the treated cell in possibly unpredictable ways. While the potential for off-target mutations has less significance in techniques such as CAR-T cell therapy, which involve somatic rather than genome edits, it is a problem that must be addressed. Many researchers, including those planning clinical trials, are using web-based algorithms to predict the off-target effects of CRISPR. However, it is now known that they are not one hundred per cent accurate, which means that off-target identifying methods will need to be greatly improved if genome editing is to be used safely to treat patients.

CRISPR has other limitations that have driven the search for alternative gene editing techniques. For example, the components of CRISPR are too large to insert into the genome of the virus normally used for gene therapy. A potential solution to this comes in the form of a mini-Cas9 that has been used successfully in mice to correct the gene responsible for muscular dystrophy. Other issues are that Cas9 will not cut everywhere it is directed to, and the pathway it uses to insert a new sequence of DNA is error-prone. Researchers are actively seeking alternative enzymes to Cas9 to expand the technique’s repertoire, in addition to strategies that involve disabling Cas9 and tethering it to other enzymes to enable different sequence changes.

In May, an entirely new gene-editing system – a bacterium-derived protein called NgAgo, which is programmed using a short DNA sequence that corresponds to the target area – caused an initial flurry of excitement. Although laboratories have so far failed to reproduce the results, the technique promises to forge a new way forward in the field.

Responsible innovation

Public concern about the use of CRISPR for genome manipulation cannot be ignored, with many people believing it paves the way to a future in which parents can choose the traits of their children. Another fear is that such germline editing enables the modified gene to be passed onto future generations with unpredictable results. Others offer more subjective arguments. Who gets to decide what constitutes an improvement to a genome? Should we be altering living organisms at all?

The scientific community fully recognises the need to tread carefully in this controversial area and to lay down specific guidelines for responsible practice. A conference in Washington DC in December 2015, to discuss the ethics of using CRISPR technology in humans, called for a “moratorium on any attempts at germline genome modification for clinical application in humans” and for a “framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome”.

It is right that scientists should continue to address public concerns and to openly discuss the benefits and risks of CRISPR. With time, it is likely that the technique will become more accepted as its extraordinary powers are realised and fears of its potential misuse are allayed. In the meantime, the fact cannot be ignored that CRISPR is already reaching into all sectors of the life sciences and it is only a matter of time before we are facing a serious technological revolution.


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These authors contributed equally: Xueli Tian and Tingxuan Gu


Basic Medical College, Zhengzhou University, 450001, Zhengzhou, Henan, China

Xueli Tian, Mee-Hyun Lee & Zigang Dong

China-US (Henan) Hormel Cancer Institute, No.127, Dongming Road, Jinshui District, 450008, Zhengzhou, Henan, China

Xueli Tian, Tingxuan Gu, Satyananda Patel, Mee-Hyun Lee & Zigang Dong

The Hormel Institute, University of Minnesota, Austin, 55912, USA

The Collaborative Innovation Center of Henan Province for Cancer Chemoprevention, Zhengzhou, China

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X.T. and M.H.L. contributed to the literature search and collection of articles, assisted with designing the figures and writing T.G. and S.P. assisted in the literature search and advised which articles were most appropriate A.M.B. edited the manuscript Z.D. supervised the studies and allocated the funding.

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