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CRISPR/CAS9 System to Treat Genetic Disorders

Paper Type: Free Essay Subject: Sciences
Wordcount: 2397 words Published: 24th Jul 2018

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An Overview of CRISPR/Cas9 and IPSCs and the Possibility of Creating a Super Force to Treat Human Genetic Disorders

  • Owen Conneran

The 14th of April 2003, the US Human Genome Research Institute completed its 15 year multibillion dollar project referred to as the Human Genome Project (HGP) and published the final sequencing map of the human genome. The ability to determine the sequencer of approximately 3 billion nucleotide base pairs that make up DNA and all the genes in DNA provided scientist with the framework to understand genetic diseases and disorders, characterized by an abnormality in one’s DNA. Research on the human genome has shown that genetic disorders can be caused by a number of factors including a mutation to one gene termed monogenic disorder; mutations to multiple genes (multifactorial inheritance disorder); or by damage to chromosome structure and/or function in part or full. These mutations can be either pass on from parent to offspring (inherited) or developed during the life of an individual. Further advancements in genomics have enabled the editing and modification of the genome by technologies which can insert, delete and modify targeted regions on a DNA sequence allowing for the control of activation or inactivation of a particular gene (Hsu et al, 2014). However, due to the colossal size of the human genome, it is extremely difficult to manipulate it. Due to the low efficiency of many techniques, a lot of scientific interest has shifted to nuclease-based genome editing techniques and in particular to the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) system. This review will discuss this platform for genome editing along with human induced pluripotent stem cells (iPSCs) and the possible combined application of both in gene therapy.


The CRISPR/Cas9 systems are loci encoded on bacterial genomes that consist of short identical, direct repeats that ranges from 21-47 nucleotide base pairs in length, interspaced with short intervening spacers derived from exogenous DNA targets called protospacers which make up the CRISPR RNA (crRNA) array (Manjunath et al, 2013). These protospacers, in each DNA target are always associated with a sequence motif termed protospacer motif (PAM) which generally vary with respect to the type of CRISPR system in question (Ran et al,2013). The CRISPR loci are surrounded by a group of CRISPR-associated (Cas) genes. The transcribed crRNA product forms a complex with a Cas protein which directs the complex to the sequence of DNA complementary to the spacer sequence. Once here, the Cas protein cuts the DNA to create a double stranded break which is then rebuilt by the cells own repair mechanisms, nonhomologous end-joining (NHEJ) or homology directed repair (HDR) (Figure 1). NHEJ is an error prone method which leaves a mark in the form of insertion or deletion (indel) mutations. Alternatively, the HDR method can be used to repair the cleaved DNA strand(s). The HDR pathway, used at lower and more variable frequencies than its counterpart NHEJ, can be used to create precise, defined modifications at a specific locus with an exogenous repair template added to the mechanism (Ran et al, 2013). This repair template can be in two forms: double-stranded DNA that targets regions with homology arms flanking the sequence to be inserted; or single-stranded DNA oligonucleotides donor sequence. Many groups have opted for this choice of repair template as it a relatively more simple method of making edits in the genome. Ran et al, also states that HDR usually only occurs in dividing cells and that the efficiency of the technique can vary greatly depending on the cell type, genomic locus of interest and the repair template used.

Fig. 1: Mechanisms’ of repair when double strand breaks (DBS) occur in the DNA. DSB caused by the CRISPR/Cas9 system can be repaired by the the processes of nonhomologous end joining (NHEJ) or homologous directed repair (HDR)/ homologous recombination (HR). These repair mechanisms can be used to alter modify the genome with the above methods of indel mutations of by repair with a desired template (far right). Figure adapted from Bassett et al, (2013).

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Type II CRISPR: The Method of Choice

Of the three types of the CRISPR system (I,II and III) identified across a large number of bacterial and archaeal hosts, the Type II CRISPR is the most well understood and best characterized and is widely used for genome editing. It contains a Cas9 nuclease, a crRNA sequence arrangement that encodes the guide RNAs and an auxiliary trans-activating crRNA (tracrRNA) that is essential in the processing of crRNA array into discrete units (Ran et al, 2013). The crRNA unit possesses a guide sequence composed of 20 nucleotides in length that directs Cas9 to a base pair target of equal length. Using the CRISPR/CAS9 system studied in the microorganism Streptococcus pyogenes as an example, it is a requirement that the DNA must precede immediately in a 5′ -NGG PAM. However, other Cas9 orthologs differ in their PAM requirements such as the Neisseria meningiditis species in which the target DNA precedes in a 5′ -NNNNGATT PAM. The RNA-guided nuclease function of CRISPR/Cas is added to mammalian cells by heterologously expressing it with human codon-optimized Cas9 and the necessary RNA constituents. In addition, a chimeric, single guide RNA (sgRNA) can be created by the fusing of crRNA and tracrRNA together. This fusion then permits Cas9 to be redirected towards virtually any DNA target in close proximity to the PAM sequence by modifying the 20 nucleotide guide sequence within the sgRNA. The Cas9 nuclease family is identified by two distinct nuclease domains, RuvC and HNH, both of which are named in accordance with homology to known nuclease domain structures (Hsu et al, 2014). HNH is a single nuclease domain whilst the RuvC domain is divided into three sub-domains. It is through the activity of these two core domains that Cas9 nucleases nick a strand of DNA to generate a double stranded break with a blunt end. Alternatively, a single stranded break (SSB) can be achieved by catalytically inactivating either of these previously mentioned domains by point mutations. A mutated form of the Cas9 enzyme known as nickase catalyses this SSB in DNA rather than the wild type enzyme which causes a double strand break in the DNA. These SSB allow for the repair of the DNA strand using the highly accurate base excision repair (BER) pathway or by HDR where the complementary strand acts as a template (Hsu et al, 2014). On the contrary, as multiplex nicking encourages the activity of NHEJ, an error prone repair mechanism, a population of cells co-targeted with a homology donor will thus have a combination of insertions and deletions (indel mutations) and donor materials.

iPSCs: Generation by Viral Transduction

IPSCs are cells that have been obtained by the nuclear reprogramming of somatic cells that share many, but not all, traits of human embryonic stem cells (hESCS). These pluripotent stem cells posses the ability of indefinite self-renewal and have the potential to differentiate into any of the various cell types in the body (Tavernier et al, 2013). In 2006, the laboratory of Shinja Yamanaka developed a method for inducing the pluripotency characteristic of stem cells in somatic cells by retrovirally inserting four transcription factors. This marked a major breakthrough in the use of stem cells as it offers a solution to the ethical issues and hampered immune responses that are associated with hESCs. In this study, Yamanaka’s team initially selected 24 genes that were believed to be critical for pluripotency in hESCs. They created retroviral vectors that encoded the cDNA of these afore mentioned genes that were introduced into an altered mouse embryonic fibroblasts (mEFs) as they contained a knock-in of a bego cassette at the Fbx15 locus which is a target gene of Oct3/4 that is expressed exclusively in mouse ESCs and early stage embryos and is thought to be critical to the maintenance of pluripotency. After Yamanka et al. transferred the 24 selected genes into the model organisms, the surviving colonies showed ESC like morphology and proliferation characteristics. These colonies were then named “induced pluripotent stem cells”. The number of genes were then reduced further until it was shown that only four genes were required to induce pluripotency. These genes are: Oct4, Sox2, c-myc and Klf-4 (Deb et al, 2009). At a later date, it was shown that this method of viral transduction also worked in human cells. However, even less genes were required for iPSCs generation, with Lin28 taking the place of c-myc and Klf4 whilst Oct4 and Sox2 remained essential (Tavernier et al, 2013). Although other methods of generating IPSCs exists, such as protein based reprogramming, Tavernier states that this viral transduction method remains amongst the most popular choice of IPSC generation due to its high efficiency.

CRISPR/Cas9, iPSCs and The Outlook

In 2013, Schwank et al. used the CRISPR/Cas9 genome editing system and stem cell technology to repair and correct the cystic fibrosis transmembrane conductor receptor (CFTR) by homologous recombination in intestinal stem cells of patients suffering from cystic fibrosis (CF), a genetic disease which mainly affects the lungs and digestive system. The team began initially by targeting the murine APC locus on adult intestinal stem cells (Schwank et al, 2013). APC functions as a negative regulator in the Wnt pathway. Inactivation of these APC alleles allowed the group to grow the stem cells in the absence of the usually required Wnt agonist R-spondin1. However, no cells grew in culture and is was later discovered that the human intestinal stem cells required further levels of Wnt for self renewal and proliferation. Stem cell organoids only grew out from the cells which had sgRNA introduced, showing the importance of the CRISPR/Cas9 system for genome editing by introducing mutations at target locations. To study the option of gene correction in adult stem cells using the CRISPR/Cas9 system, the group focused on the CTFR in intestinal stem cells, mutations of which cause CF. Schwank states that the two CF patients used in this study both had a deletion of phenylalanine at position 508 in exon 11, resulting in premature CTFR degradation. The patients organoids were then independently transfected with two sgRNAs that targeted the CTFR exon 11 or intron 11, coupled with the wild type CTFR embedded in a donor plasmid. The function of the sgRNAs was to make a genomic cut on the CTFR sequence within the target vector. Multiple organoid clones with each type of the sgRNA were recovered after transfection and site specific knock-in events and amendment of the F508 del allele were observed by sequencing the recombined allele which caused heterozygous repair in the majority of the clones. Using another sgRNA (sgRNA2), that caused a DSB 203 bps further down of the F508 del mutation resulted in a clone that also produced knock in effects but downstream of the mutation and repair was not accomplished. To clarify and prove that the function of the CTFR had been restored, Schwank carried out a forskolin assay with trangenic cell lines that demonstrated large expansion of the surface area of the organoid compared to no expansion of surface area in control organoids that were untransfected. They then examined whether these forskolin induced expansion of the amended organoids was sensitive to chemical inhibition of CFTR by CFTRinh-172 which has been suggested to imitate the inflammatory profile of CF. As expected, these swelling were fully destroyed in the presence of the inhibitor and showed that the F508 del mutation had resumed its function and maintained the CFTR phenyotpe in the studied organoids.

Fig 2: Overview of gene correction in CF patients. Stem cells, shown in green, are the only cell type capable of integration and that become new organoids. Adapted from Schwank et al, (2013).

The results obtained from this study provide a promising outlooks for the CRISPR/Cas9 genome editing system and in turn for its use as a treatment for genetic disorders. Although adult stem cells were used as opposed to IPSCs, this method may present a platform for IPSC based approaches in future gene therapy experiments and quite possibly, IPSCs may be the one cell to rule them all.


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Schwank G, Koo B.K, Sasselli V, Dekkers J.F, Heo I, Demircan T, Sasaki N, Boymans S, Cuppen E, Van der Ent C.K, Nieuwenhuis E.E.S, Beekman J.M and Clevers H (2013) Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients. Cell Stem Cell. 13:653-658

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