Clustered regularly interspaced short palindromic repeats (CRISPR) along with CRISPR-associated protein 9 (Cas9) are an acquired immune system found in many prokaryotes (Jinek et al., 2012). Immunity based on the CRISPR-Cas9 complex uses guide RNA (gRNA) to direct a Cas9 nuclease to the target site and cleave invading DNA (Figure 1) (Jiang and Doudna, 2017). This prokaryotic system has now been adopted by researchers as a gene editing tool (Zhang et al., 2014). Many researchers are particularly interested in using CRISPR to treat and cure genetic diseases. When a synthetic gRNA attached to Cas9 recognizes a specific sequence of the genome a double strand break is made. Cellular repair mechanisms repair the DNA break thereby removing the disease-causing mutation (Zhang et al., 2014) (Figure 2). Genetic diseases lead to the premature death of those who inherit the underlying gene mutations, so a gene editing tool such as CRISPR has the potential to save lives if the technology can be applied to living patients. Despite its promises, there are some barriers holding CRISPR-Cas9 back from its full therapeutic potential. One of which is its ability to induce off-target mutations (Zhang et al., 2015). This is when Cas9 makes cuts at sites that are similar to the target sequence (Jiang and Doudna, 2017). This essay will explore two techniques that are currently being used to combat the issue of off-target effects. Finally, a successful application of CRISPR-Cas9 to treat a genetic disease will be discussed.
Although gRNA is made to target a specific sequence, researchers have found that the CRISPR gene editing system can cause unintended mutations known as ‘off-target’ effects. (Zhang et al., 2015). The gRNA might bind to a region with three to five mismatched base-pairs, thereby causing unwanted DNA breaks (Zhang et al., 2015). In a recent study, CRISPR-Cas9 was successfully used to restore sight in mice with a mutated Pde6b gene; however, whole genome sequencing of two CRISPR-Cas9 treated mice revealed 117 insertions and deletions, and 1,397 single nucleotide changes were identical in both mice (Schaefer et al., 2017). Reports such as these currently preclude the use of CRISPR-Cas9 mediated gene editing in clinical settings; however, research into the use of CRISPR for gene editing only started in 2012 and researchers are working on techniques to combat the challenge of off-target mutations (Jinek et al., 2012, Zhang et al., 2015). One method currently being investigated is the use of modified Cas9 nucleases (Kleinstiver et al., 2016). The study utilized a variant of the commonly used Streptococcus pyogenes Cas9 (spCas9) called SpCas9-High-fidelity variant 1 (HF1), and its activity in human tissue samples was tested (Kleinstiver et al., 2016). The variant was made by altering four DNA contact points on the Cas9 protein. And then genome wide unbiased identification of double-strand breaks enabled by sequencing (GUIDE-seq) was used to search for off-target effects caused by SpCas9-HF1 (Figure 3) (Kleinstevir et al., 2016). GUIDE-seq revealed that off-target mutations were undetectable when SpCas9-HF1 was used (Kleinstiver et al., 2016). This shows that by making relevant changes to the Cas9 nuclease its specificity can be improved. Thereby opening up the potential for CRISPR-spCas9-HF1 to be used in clinical settings.
Not only can variants of Cas9 protein be used to improve the specificity of gene editing, but researchers are also working on identifying CRISPR systems in other prokaryotes (Shmakov et al., 2017). For a gene to be cleaved by Cas9, a Protospace Adjacent Motif (PAM sequence) must be downstream of it. SpCas9 recognizes the PAM DNA base sequence 5’-NGG-3’. (Nakade et al., 2017). However, the sequence may not be in the correct position to target the gene of interest. Due to this limitation, researchers are searching for new CRISPR systems in other bacteria (Nakade et al., 2017). One newly characterized nuclease is called Cpf1 (Zetsche et al., 2015). In a study carried out by Zetsche et al. (2015), they explored the efficiency of Cpf1 for gene editing in human cells and found that the Cpf1 nuclease from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) could edit genes in human cells successfully. The gRNA used by Cpf1 is even simpler than that of Cas9, so this could simplify the process of designing gRNA and reduce cost. Additionally, they discovered that unlike Cas9 that produces blunt ends, Cpf1 produces cohesive ends with 5’ overhangs. This could facilitate DNA repair via non-homologous end joining (NHEJ) (Figure 2) (Zetsche et al., 2015). AsCpf1 and LbCpf1 nucleases were found to be highly specific in human cells, showing no detectable off-target effects (Kleinstiver et al., 2016). As more nucleases are characterized from other prokaryotic species the regions that can be targeted will increase, and the specificity of gene editing will improve.
CRISPR mediated gene editing is not yet perfected; however, several studies have reported cases of successful treatment of genetic diseases using CRISPR. In a recent study conducted by Wen et al. (2017), they attempted to use CRISPR to treat Sickle Cell Disease (SCD). SCD is a blood disorder caused by a single base pair change in the hemoglobin beta chain gene (HBB) that causes abnormal hemoglobin S (HbS) to be made. If an individual is homozygous for the sickle cell HbS mutation they will suffer from SCD (Wen et al., 2017). Thus, in an attempt to repair this mutation, hematopoietic stem cells were collected from a patient with SCD and treated using CRISPR-Cas9. These are the stem cells that differentiate into other blood cells. They reported that the edited cells became heterozygous for the HbS allele, such that they no longer had SCD. They also performed cell sickling assays that showed that the normal function of the red blood cells was restored. In such early stages of CRISPR use, results such as this reveal the potential of the technology.
CRISPR has the potential to eradicate genetic diseases as long as certain issues are resolved. One of which is off-target mutations. To solve this issue some researchers are making alterations to the Cas9 nuclease in an effort to increase its specificity. Meanwhile, others are studying the CRISPR systems found in other prokaryotes. Research into the use of CRISPR for gene editing began 5 years ago and huge advances have already been made. This progress suggests that CRISPR has the potential to help with the eradication of genetic within my lifetime.
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