Essay: CRISPR Cas9: A Revolutionary Genomic Editing Tool

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  • CRISPR Cas9: A Revolutionary Genomic Editing Tool
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The ability to engineer genomic DNA in cells and organisms easily and precisely will have major implications for basic biology research, gene therapy, biotechnology and the future of medicine. Technologies for making and manipulating DNA have enabled many of the advances in biology over the past 60 years1. The introduction of genomic sequencing technologies and the generation of whole-genome sequencing data for large numbers and types of organisms has been one of the most important advances of the past two decades regarding genome science. The CRISPR-Cas9 system has proven difficult to use in the lab however, the implications of the technology may change the face of genome science and genetic diseases as we know it.

Since the discovery of the DNA structure by Watson and Crick in 1953, researchers have been searching for ways to make site-specific changes to genomes. The RNA-guided enzyme Cas9, which originates from the CRISPR-Cas adaptive bacterial immune system, is transforming biology by providing a genome engineering tool based on the principles of Watson-Crick base pairing1. The application of CRISPR technology in genome-wide association studies will enable large-scale screening for drug targets, SNPs and potential SNP repair as well as other phenotypes and will facilitate the generation of engineered animal models that will benefit pharmacological studies and the understanding of human diseases among many other things1.

CRISPRs or clustered regularly interspaced palindromic repeats were first described in 1987 by Japanese researchers as a series of short direct repeats interspaced with short sequences in the genome of Escherichia coli2. It wasn’t until 2002 that CRISPRs were found to be numerous in bacteria and archaea at which point they were predicted to play a role in DNA repair3. Upon more investigation into CRISPR loci it was found that they are transcribed and that Cas – which are CRISPR-associated genes – encode proteins with putative nuclease and helicase domains4.

It was soon after proposed that CRISPR-Cas is an adaptive defense system against invading phages and plasmids which functions analogously to the eukaryotic RNA interference (RNAi) systems5. In 2008, mature CRISPR RNAs (crRNAs) were shown to serve as guides which complex with Cas proteins to interfere with virus proliferation in E. coli6. Concurrently in 2008, CRISPR-Cas was harnessed for its DNA targeting activity in Staphylococcus epidermidis7.

Functional CRISPR-Cas loci are comprised of a CRISPR array of identical repeats intercalated with invader DNA-targeting spacers that encode the crRNA components and an operon of Cas genes encoding the Cas protein components8. The protospacer adjacent motif (PAM), a short sequence motif adjacent to the crRNA-targeted sequence on the invading DNA, plays an essential role in the stages of adaptation and interference in CRISPR systems8. In naturally occurring environments, viruses can be matched to their bacterial or archaeal hosts by investigating CRISPR spacers. Experiments in this area have shown that viruses are constantly evolving to avoid CRISPR-mediated attentuation8.

In 2012, the S. pyogenes CRISPR-Cas9 protein was shown to be a dual-RNA–guided DNA endonuclease that uses the tracrRNA:crRNA duplex to direct DNA cleavage9. Trans-activating crRNA (tracrRNA) is a small RNA that is trans-encoded upstream of the CRISPR-Cas locus in Streptococcus pyogenes and has been reported to be essential for crRNA maturation9.

Cas9 uses an HNH endonuclease domain to cleave the DNA strand that is complementary to the 20-nucleotide sequence of the crRNA. Another separate but similar endonuclease domain RUVc of Cas9 cleaves the DNA strand opposite the complementary strand8. Mutating either domain in Cas9 generates a variant protein with single-stranded DNA cleavage (nickase) activity, whereas mutating both domains results in an RNA-guided DNA binding protein9. The RNA-guided CRISPR-Cas9 system will cause a double stranded break, unless otherwise mutated to form a nickase, in the DNA at which point the cell’s native double stranded break repair mechanisms will repair the DNA in hopes that the proper sequence is reconfigured.

The dual tracrRNA:crRNA was subsequently engineered as a single guide RNA that retains two critical features: the 20-nucleotide sequence at the 5′ end of the RNA that determines the DNA target site by Watson-Crick base pairing, and the double-stranded structure at the 3′ side of the guide sequence that binds to Cas99. Mechanistic studies also show that the PAM is critical for initial DNA binding and in the absence of the PAM, even target sequences fully complementary to the guide RNA sequence are not recognized by Cas99. This finding revealed a simple two-component system in which changes to the guide sequence (20 nucleotides in the native RNA) of the RNA can be used to program CRISPR-Cas9 to target any DNA sequence of interest as long as it is adjacent to a PAM9.

In contrast to other genomic targeting and editing technologies like TALENs or ZFNs which require substantial protein engineering for each DNA target site to be modified, the CRISPR-Cas9 system requires only a change in the guide RNA sequence to target your sequence of interest9. For this reason, the CRISPR-Cas9 technology using the S. pyogenes system has been rapidly and widely adopted by the scientific community to target, edit, or modify the genomes of a vast array of cells and organisms8. Due to its high practicality and versatility I foresee CRISPR-Cas9 to be integral to much genomic research throughout the scientific community.

CRISPR-Cas9 Mechanism of Action

Structural analysis of S. pyogenes Cas9 has revealed insights into the mechanism of CRISPR-Cas9. Molecular structures of Cas9 determined by electron microscopy and x-ray crystallography show that the protein undergoes large conformational rearrangement upon binding to the guide RNA, with a further change upon association with a target double-stranded DNA10. The conformational change creates a channel which runs between the two structural portions of the Cas9 protein, that binds to the RNA-DNA hybrid as well as to the coaxially stacked dual-RNA structure of the guide corresponding to the crRNA repeat–tracrRNA anti-repeat interaction10. The conformational change in Cas9 is speculated be part of the mechanism of target dsDNA unwinding and guide RNA strand invasion, although this idea remains to be tested8.

The Cas9 protein remains inactive in the absence of guide RNA9. This characteristic makes of Cas9 makes for a great control for experiments using the system. One can utilize an active Cas9 with a guide RNA in conjunction with an inactive control which lacks a guide RNA. Once the Cas9 protein is activated, it stochastically searches for target DNA by binding with sequences that match its PAM sequence9. The PAM in Streptococcus pyogenes is 5′-NGG-3′9. When the Cas9 protein finds a potential target sequence with the appropriate PAM, the protein will melt the bases immediately upstream of the PAM and pair them with the complementary region on the guide RNA9. If the complementary region and the target region pair properly, the RuvC and HNH nuclease domains will cut the target DNA after the third nucleotide base upstream of the PAM.

To assess the target-binding behavior of Cas9 in cells, researchers used chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) to determine the numbers and types of Cas9 binding sites on the chromosome11. Results showed that in both human embryonic kidney (HEK293) cells and mouse embryonic stem cells (mESCs), a catalytically inactive version of Cas9 bound to many more sites than those matching the sequence of the single guide RNA used in each case11. Off-target interactions could lead to potential problems in the system which include improper DNA cleavage at non-specific sites.

Off-target in
teractions with DNA such as these are typically at sites bearing a PAM and partially complementary to the guide RNA sequence11. These interactions are consistent with established modes of DNA interrogation by Cas9. Interestingly, active Cas9 rarely cleaves the DNA at off-target binding sites, implying binding and cleavage events are not coupled and nearly perfect complementarity between the guide RNA and the target site are necessary for efficient DNA cleavage11. I, however, am still weary when it comes to off target DNA interactions. I believe the mechanism of action must be completely ironed out, meaning we need to understand what exactly happens when Cas9 binds to alternative sites. Once we believe beyond a reasonable doubt that perfect complementarity is needed, then we can deem is safe for use in humans.

CRISPR-Cas9 is an Efficient Tool to Modify Genomes

Since its demonstration as a genome editing tool in Arabidopsis thaliana and Nicotiana benthamiana, editing has been demonstrated in a variety of crop plants including rice, wheat, and sorghum as well as sweet orange and liverwort8. This technology has the potential to change the pace and course of agricultural research. For example, a recent study in rice found that target genes were edited in nearly 50% of the embryogenic cells that received the Cas9 guide RNA constructs, and editing occurred before the first cell division12. These genetic changes were passed to the next generation of plants without new mutation or reversion, and whole-genome sequencing did not reveal substantial off-target editing12. Such findings suggest that modification of plant genomes to provide protection from disease and resistance to pests may be much easier than has been the case with other technologies12. Useful and genetic changes to crops may unsure their survival in the face of invasive pests and climate change rendering CRISPR-Cas9 a near necessity in the coming years.

In a different highly influential study, the CRISPR-Cas9 system was used to modify the genome of intestinal stem cells derived from patients with the cystic fibrosis transmembrane conductor receptor (CFTR) mutation. CFTR encodes an ion channel essential for fluid and electrolyte homeostasis of epithelia and a point mutation and single amino acid change results in cystic fibrosis. Studies have shown successful transplantation of clonal organoids derived from single Lgr5+ stem cells into damaged tissue for mouse colon and liver, making the organoid system a promising tool for adult stem cell therapy and gene therapy13. Thus, the idea of culturing organoids with the CFTR mutation followed by CRISPR-Cas9 editing was born.

To investigate the possibility of gene correction in adult stem cells using CRISPR-Cas9, the investigators focused on the cystic fibrosis transmembrane conductor receptor in primary cultured small intestinal and large intestinal stem cells. When the organoids containing the mutation were mature they transfected the patient organoids with single guide RNAs for CFTR exon 11, where the CFTR mutation is located, together with a donor plasmid encoding wild-type CFTR sequences13. The researchers then confirmed site-specific knock-in events and correction of the F508 del allele by sequencing the recombined allele13. The researchers went on to confirm that the CRISPR-Cas9 system did not affect off-target sites, and therefore demonstrated high specificity in adult stem cells.

After confirming that the cystic fibrosis mutation had been reversed in the organoids they went on to determine whether the DNA sequence rendered a functional ion channel. They measured swelling of organoids as a surrogate for a functioning ion channel and found that the organoids swelled to a similar degree to wild-type organoids13. Lastly, they added a CFTR inhibitor to the CRISPR modified patient organoids and found that swelling was fully abolished demonstrating that the corrected F508 del allele was fully functional and was able to rescue the CFTR phenotype in organoids via the CRISPR-Cas9 system. Together with previous studies, in which in vitro expanded organoids were successfully transplanted into colons of mice, this work provides a potential strategy for future gene therapy in patients13. This application of CRISPR-Cas9 is one type of a number of ground breaking genetic experiments possible using this system. I predict we will see CRISPR being used in labs around the world in order to attack genetic diseases similar to Cystic fibrosis.

The CRISPR-Cas9 genome editing system has potentially even greater implications in cancer research. Certain forms of cancer such as acute myeloid leukemia and Ewing’s sarcoma are a result of chromosomal translocations. To gain insight into these cancers a reliable model is necessary in order to study the diseases. One group of researchers used the CRISPR-Cas9 system to engineer double stranded breaks and produce chromosomal translocations in human cell lines to induce these cancers in vitro14. Using CRISPR-Cas9 the researchers were able to reproduce the chromosomal translocations necessary in acute myeloid leukemia and Ewing’s sarcoma. In addition, they were able to produce the functional fusion gene and gene products in both cases14. The frequencies obtained indicate that this is a robust and efficient strategy for modelling the effects of rearrangements produced by chance in cancer cells14. Though this did not directly affect an organism in vivo, the researchers outlined a reliable model in order to study these cancers. Models like these are born due to the use of CRISPR-Cas9. Continuing the development of different models and strategies may eventually lead to in vivo remedies for such cancers.

Another interesting example of the potential use of the CRISPR-Cas9 system is exemplified by an experiment in C. elegans. Researchers harnessed the CRISPR-Cas9 system to introduce mutations to the unc-119 and dpy-13 genes in the germ cells of wild type worms15. These genes were targeted because loss-of-function of these genes cause phenotypic amorphisms that are easily identifiable15. Once the genes were introduced into the worms the F1 progeny were analyzed for the mutations and were confirmed to possess the mutation. The same was true for the F2 worms15. The researchers then introduced a single guide RNA to the F2 generation such that the CRISPR system would repair the mechanism and rescue the phenotype. The phenotype was rescued in the two subsequent generations following the F2 progeny15. These results suggest the potential of the CRISPR-Cas9 system in heritable genome editing in a wide variety of multicellular eukaryotes15.

Outlined here are just three practices each of which harness the unique versatility of the CRISPR-Cas9 system in different ways. These are only a few of a myriad of potential uses of this system regarding medicine and public health. The future beholds many more labs exploiting the power of CRISPR-Cas9 which will undoubtedly lead to great scientific progress.

Drawbacks of the CRISPR-Cas9 System

Despite the great potential of CRISPR-Cas9 in genome editing, there are some important issues that need to be addressed. Some of these issues include off-target mutations briefly mentioned above, PAM dependence, and delivery methods of CRISPR-Cas9. Organisms with large genomes like plants often contain multiple DNA sequences that are identical or highly homologous to target DNA sequences. Besides target DNA sequences CRISPR-Cas9 also cleaves these identical or highly homologous off-target DNA sequences, which leads to mutations at undesired sites, called off-target mutations16. Off target mutations can lead to numerous undesired effects dependent on where the homologous sequence is located on a chromosome.

The amount of Cas9 enzyme expressed in the cell is another important factor in tolerance to mismatches. High concentrations of the enzyme were reported to increase off-site targeting, whereas lowering the concentration of Cas9 increases specificity while
diminishing on-target cleavage activity16. Thus, a delicate balance needs to be achieved in order to maximize the potential of the CRISPR-Cas9 system. This balance is something many lab technicians have struggled with when utilizing the system16.

Aside from guide RNA/target sequence complementarity the CRISPR-Cas9 system is also dependent on 2-5 nucleotide PAM sequence immediately downstream of the target sequence16. Since this is the case, the PAM-dependent manner of CRISPR-Cas9-mediated DNA cleavage constrains the frequencies of targetable sites in genomes16. This problem can be slightly optimized by using different Cas9 orthologs from various other bacteria in which the PAM sequence is different and possibly more abundant in your organism of interest.

Questions also remain regarding the delivery methods of CRISPR-Cas9 into organisms. DNA and RNA injection-based techniques are used for CRISPR-Cas9 delivery, such as injection of plasmids expressing Cas9 and guide RNA and injection of CRISPR components as RNA16. The efficiencies of delivery methods depend on the types of target cells and tissues and may prove difficult if the long term-goal is human use16.

With regard to the implementation of CRISPR-Cas9 in plants, a low frequency of germline genome editing by CRISPR-Cas9 is observed in certain plants, particularly the model organism Arabidopsis17. When Arabidopsis plants are subjected to Agrobacterium transformation with CRISPR-Cas9 by floral dip, mutation efficiency of target sites can be high in somatic cells, but can be low in reproductive cells, limiting the likelihood that mutations will be inherited in the next generation17. However, it is possible to promoters to express Cas9 in germline-specific cells to generate higher frequency of mutations in subsequent generations in Arabidopsis.

Potential uses for the CRISPR-Cas9 System beyond Genome Editing

A group of researchers engineered a catalytically deactivated version of Cas9 (dCas9) which has been repurposed for targeted gene regulation on a genome-wide scale18. Referred to as CRISPR interference (CRISPRi), this strategy was shown to block transcriptional elongation, RNA polymerase binding, or transcription factor binding, depending on the site(s) recognized by the dCas9–guide RNA complex18. Demonstrated first in E. coli, whole-genome sequencing showed that there were no detectable off-target effects. CRISPRi has been used to repress multiple target genes simultaneously, and its effects are reversible. RNA-seq analysis showed that CRISPRi-directed transcriptional repression is highly specific18.

More broadly, these results demonstrated that dCas9 can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence in a genome, laying the foundation for future experiments involving genome-wide screening similar to those performed using RNAi18. The lack of CRISPR-Cas systems in eukaryotes is an important advantage of CRISPRi over RNAi for various applications in which competition with the endogenous pathways is problematic.

In addition to CRISPRi experiments, CRISPR-Cas9 can also be used to engineer epigenetic modifications. Fusing dCas9 to p300, a mammalian acetyltransferase, for example can induce the acetylation of histone H3 at lysine 2719. This mark opens associated DNA which will lead to increased gene activation19. However, gene activation or inactivation is only the surface of this technology which could include dCas9 fused to a variety of histone post-translational effectors. It is within the realm of possibility that dCas9 fused to domains that can control deacetylation, methylation, or phosphorylation of histones could also be of use to influence gene expression in eukaryotic cells19. As one can imagine, this type of application of CRISPR-Cas9 can be used in a multitude of different experiments involving gene expression and silencing.

A future direction of this tool that is currently attainable would be its application in conjunction with genome-wide association studies (GWAS). Such studies uncover SNPs that may be associated with particular diseases. GWAS studies have already uncovered many specific SNPs that are known to correlate with the onset and cause of certain diseases. CRISPR-Cas9 makes it possible to repair those particular SNPs in the genome and thus treat disease20. Therefore, CRISPR-Cas9 in combination with high throughput sequencing may be able to prevent disease in certain circumstances. This type of approach has major implications with regard to populations with a known history of a particular disease and its associated SNPs20. If they are able to be reverted using CRISPR-Cas9, and this is able to be transferred through generations, it may be possible to reverse diseases plaguing susceptible populations.

I believe once the mechanism and components of the CRISPR-Cas9 system are fully understood the system will become a household name. A couple questions that need to be answered regarding future improvements include: is there a way to harness this system without the use of a PAM sequence? Additionally, can we eliminate off-target attacks on DNA in vivo and make targeting near perfect? Once questions like these are answered we will be well on our way to making land breaking progress in the world of genome science.


There are many other potential uses and results that have been left out of this review of the CRISPR-Cas9 system. However, this paper provided insights into the simple two-component CRISPR-Cas9 system, using Watson-Crick base pairing by a guide RNA to identify target DNA sequences. There are many examples of how CRISPR-Cas9 is a versatile technology that has already stimulated innovative applications in biology and biotechnology. Furthermore, specific methods for delivering Cas9 and its guide RNA to cells and tissues should benefit the field of human gene therapy. For example, recent experiments confirmed that the Cas9 protein-RNA complex can be introduced directly into cells using nucleofection or cell-penetrating peptides to enable rapid and timed editing22. Understanding the specifics of repair after Cas9-mediated DNA cutting will advance the field by enabling efficient insertion of new or corrected sequences into cells and organisms.

In general, the lack of efficient, inexpensive, fast-to-design, and easy-to-use precision genetic tools has also been a limiting factor for the analysis of gene functions in model organisms of developmental and regenerative biology. This road-block is beginning to be removed with the onset of CRISPR-Cas9. Efficient genome engineering to allow targeted genome modifications in the germ lines of animal models such as fruit flies, mice, rats, and nematodes, is now possible with the development of the CRISPR-Cas9 technology. The technology can also facilitate the generation of mouse and rat models better suited to pharmacological studies and the understanding of human diseases22.

Overall, CRISPR-Cas9 is already having a major impact on functional genomic experiments that can be conducted in various model systems, which will advance the field of experimental biology in ways not imagined even a few years ago. Numerous examples were provided that shined light on the potential the CRISPR-Cas9 technology has in the world of genomic science. From optimizing crop yields of rice, wheat and sorghum, to the repair of an ion channel responsible for the inherited disease of cystic fibrosis to the potential of generational repair of recessive alleles in vivo, the CRISPR-Cas9 system may truly change the face of biology and medicine. The system is not without its flaws, but based on what has been reported, the system can be optimized to perform the way the scientific community hopes that it will.


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