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Gene Editing:
ARK believes the progress in gene editing is astonishing and holds the promise of curing many diseases including cystic fibrosis, sickle cell disease, beta-thalassemia, tay sachs and many others. However, the differentiation between gene editing technologies and which will be best suited to cure which disease is less well-understood.
During the past decade, the concept of editing genes to potentially cure genetic disorders has received a lot of attention, especially in academic circles. After 2014, CRISPR-Cas9 began to establish itself as a clear differentiator in gene editing, as opposed to other methods, like transcription activator-like effector nucleases (TALEN), its predecessor, that was the state of the art prior to CRISPR-Cas9 (Figure 1).
Figure 1.
*Certain data included herein are derived from Clarivate Web of Science. © Copyright Clarivate 2021. All rights reserved.
CRISPR-Cas proteins are proficient at going to a specific DNA sequence and disrupting genes. Scientists discovered that CRISPR-Cas9 was a novel and inexpensive way to target a DNA sequence. However, these proteins create double-strand DNA breaks, which cause uncontrolled mixtures of “indels”, insertions and deletions, as well as cell changes like translocations, p53 activation, chromothripsis, and other large deletions at the target site.
Naturally, scientists have been trying to build additional molecular tools that can enable precise gene correction rather than gene disruption. In this review, we outline the different technologies and their potential use cases.
CRISPR Cas9 or Cas12a:
Clustered Regular Interspaced Short Palindromic Repeats (CRISPR Cas9 or Cas12a) are a cost-effective, simple, and extremely useful gene-editing tool.
CRISPR-Cas9 proteins act like molecular scissors that cut DNA at target sites to damage target genes and potentially alleviate genetic diseases that can be treated by disrupting a gene (Figures 2 & 3). The editing system is composed of two parts, its enzyme, which could be Cas9 or others, and the guide RNA. The guide RNA brings the Cas protein to the target portion of DNA that a scientist wants to manipulate, and the enzyme cuts the double-stranded DNA. To repair the cut DNA, the cell uses a natural repair mechanism called “end joining”.
Rejoining the cut DNA perfectly regenerates the starting sequence, with no change in the DNA. But during the course of end-joining mistakes are common, and the result is an indel, which usually results in gene disruption. Scientists are concerned that gene editing with CRISPR enzymes may cause unintended consequences to cells because double-strand DNA cuts are known to trigger many cellular emergency responses that may leave cells in an unhealthy or even cancerous state.
However, CRISPR-Cas9 proteins have shown promise to treat diseases of high unmet need, including diseases that can be treated by stopping the production of a protein, and those that can be treated by disrupting one gene to reactivate a healthy gene. For example, CRISPR-Cas9 has shown clinical promise to treat transthyretin amyloidosis (ATTR), a condition where there is an excess buildup of amyloid protein in the lung and heart. By using CRISPR-Cas9 scissors to disrupt the transthyretin (TTR) gene, the protein production stops, thus ameliorating the disease.
In a second instance, CRISPR-Cas9 may be helpful for disrupting a gene to reactivate another one. For example, in sickle cell disease and beta-thalassemia, which are blood disorders, by damaging genes that silence the healthy fetal hemoglobin gene (HBG), CRISPR-Cas9 has shown clinical promise reactivating the production of fetal hemoglobin (HbF). The reactivated HbF can compensate for mutated adult hemoglobin genes (HBB), providing healthy hemoglobin.
Figure 2.
Figure 3.
Base Editing:
Base editing (BE), a technology that emerged from Dr. David Liu’s labs at the Broad Institute, Harvard University, and Howard Hughes Medical Institute (HHMI) decreases the possibility of off-target edits due to higher precision editing and the avoidance of double-strand DNA cuts.
Base editors nick one strand of DNA but do not make double-stranded DNA breaks. The Cas9 domain in a base editor has one nuclease domain inactivated, which is why only one strand of the target DNA is nicked, as opposed to Cas9, which has two active nuclease domains and cuts both strands of the DNA. Cells naturally experience thousands of nicks every day, but only rarely experience double-strand DNA cuts.
CRISPR-Cas9 can effectively only disrupt a gene, but BE could selectively disable *or* enable a specific genetic function by correcting a single ‘misspelling’ in the genome.
BE is composed of three parts, as opposed to CRISPR-Cas9 which is composed of two. In BE, the inactivated Cas9 domain brings a deaminase enzyme to its target location so that it can convert one DNA base into a different base (Figure 4 & 5). The deaminase converts the target base (C to T, or A to G), and the nickase activity of the base editor nicks the non-edited strand to stimulate the cell into correcting the other strand as well.
BE is comparable to a pencil and eraser (Figure 6), you may get some lead or eraser debris, but your paper will likely look better than it did in the first instance when you cut it and tried to crudely put it back together again. With the pencil and eraser, you can correct mutated DNA letters that cause thousands of genetic diseases.
Figure 4.
Figure 5.
*3D printed model of a base edit.
Figure 6.
Prime editing:
Prime editing (PE), the latest editing technology to emerge from Dr. David Liu’s labs at the Broad Institute, Harvard University, and HHMI, adds new functionality to the CRISPR-Cas9 protein. Thanks to this additional utility, PE could treat many more rare and complex diseases.
PE is composed of four parts; a primer binding sequence and a reverse transcriptase are added to write DNA from an RNA template.
In living cells, PE can copy parts of a DNA sequence into a targeted DNA site, without cutting the DNA double helix, which causes unintended consequences to the genome. Like BE, its predecessor, PE avoids these undesired outcomes.
A prime editor involves a disabled CRISPR-Cas9 protein — which cannot make double-strand DNA breaks but can still be targeted by a guide RNA — attached to an engineered enzyme known as reverse transcriptase. Transcription describes the process by which a DNA template produces RNA, while a reverse transcriptase does the opposite — writing DNA with an RNA template.
In PE, the “Prime Editor Guide RNA” (pegRNA), both guides the Cas9 protein to the correct DNA sequence and specifies what part of the edited sequence should be inserted into the target site. There, the disabled Cas9 protein nicks one strand of the DNA (Figure 7). The engineered reverse transcriptase, which is fused to the Cas9 protein, copies the sequence containing the desired edit from an extension in the pegRNA into the cell host’s DNA at the freshly nicked target site. The cell incorporates the newly copied DNA sequence into its genome, resulting in a permanent change.
Figure 7.
Because researchers can specify virtually any sequence with dozens of nucleotides to replace the starting sequence in the genome, PE has been compared to a word processor, which is more precise and versatile than editing with CRISPR-Cas9, or molecular scissors (Figure 8). The latter disrupts rather than fixes genes but does not correct them and based on early data, PE may be less prone to off-target editing than CRISPR-Cas9 alone (Figure 9).
Figure 8.
Figure 9.
Source: https://www.nature.com/articles/s41586-019-1711-4
Although these technologies are very exciting, their success is possibly contingent on the original discovery and inclusion of a CRISPR Cas protein. Furthermore, CRISPR-Cas9 has been used in humans (successfully!), whereas others are still in pre-clinical testing.
ARK believes that these technologies will be part of an ever-growing gene-editing toolbox and will have important roles in different indications. With gene editing, innovations will continue to proliferate and cross-licensing among technologies is a likely outcome (Figure 10).
Figure 10.
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