There are a wide variety of diseases that affect humans. Some are infectious and some genetic. When people think of genetic diseases they typically think of whole missing genes. However, even just a single base change out of our over three billion nucleotide genome can result in devastating and unfortunately rather common genetic diseases. 1 in 7,000 males worldwide are affected by muscular dystrophy, 1 in 12 people are carriers for Cystic Fibrosis, and 1 in every 365 African Americans has Sickle Cell Anemia. Each of these diseases can arise from a single nucleotide change.

CRISPR

In the last few years the CRISPR/Cas9 system has revolutionized what is possible in genome editing. Using this system any area of the genome can be targeted and edited.

Flaws

However, the technology is not perfect due to its reliance on blunt end joining. There are often errors that happen when the DNA strands join back together. Because the normal CRISPR systems edits DNA, these errors are permanent.

A paper came out May of this year investigating performing single base nucleotide edits in double stranded DNA without any strand breaks. They did this through the use of the editing enzyme APOBEC1, which performs C to U edits. However there were still some issues with this system. There was only a 50% chance of their edited strand getting chosen and the desired edit happening. Also they could only perform a C to U edit, so the capabilities were limited.

Targeting RNA

Based of of this we started to think about editing the mRNA. But is targeting the RNA with CRISPR/Cas9 possible?

A paper published in 2014 showed that it is possible to target RNA with CRISPR through the use of a PAMmer. This is a short oligonucleotide that is separate from Cas9 but helps to guide the system there and activates nuclease which performs the cleavage. Through the use of a PAMmer, dCas9 was also showen to be able to target mRNA.

Advantages

There are many advantages to targeting mRNA. If any mistakes happen, they won't be permanent because the DNA is not being edited. The system will be regulated, and we can double the edits possible. Not only will we be able to use APOBEC1 and perform C to U edits, but also ADAR which performs A to I edits exclusively in RNA. I, Inosine, is a nucleotide that is common in brain pathways and is read by the ribosome as a G.

VV this stuff will be gone VV

Background

Previous research on the CRISPR-Cas9 system has shown that Cas9 can cleave single-stranded RNA with the help of PAMmer (O’Connell et al., 2014), and RNA-targeting Cas9 (RCas9) can track mRNA in living cells (Nelles et al., 2016). The enzymatically inactive form of Cas9, dCas9, can also target specific sequences and act as a guide for a base-editing enzyme (Komor et al., 2016). These studies serve as the basis of our experimental design.

CRISPR-associated protein 9 (Cas9) is an endonuclease that has been shown to be able to cleave double-stranded DNA at designated sites guided by an RNA sequence (guide RNA) complementary to the target site. The activation of Cas9 depends on the recognition of the protospacer adjacent motif (PAM), which is a short sequence next to the targeting site on the opposite strand. For targeting single-stranded RNA, a PAMmer (PAM-presenting oglionucleotide) is required to stimulate the activity of Cas9 (O’Connell et al., 2014).

APOBECs are a group of cytidine deaminases found in a range of mammals, with several variants identified. These enzymes, as the name indicates, can perform cytidine to uracil base editing on nucleotide sequences by deaminating the cytidine. The APOBEC1 gene is located on chromosome 12 in humans, and the enzyme coded by this naturally targets mRNAs. Experiments have shown that APOBEC can also edit DNA sequences using the CRISPR-Cas9 system (Komor et al., 2016). The said experiment used an APOBEC1-XTEN-dCas9 system, in which APOBEC is joined to dCas9 with an XTEN linker that limits the range that can be reached by the enzyme.

ADARs are a group of adenosine deaminases that act specifically on RNA. They can perform adenosine to inosine single base editing. ADAR1 and ADAR2 are the two catalytically active variants in mammals. Both contain a deaminase domain and several dsRNA binding domains, while ADAR1 has two Z-DNA binding domains as well (Savva et al., 2012). For the purpose of our project, only the deaminase domains were selected for cloning.

Motivation

There are a wide variety of diseases that affect humans. Some are infectious and some genetic. When people think of genetic diseases they typically think of whole missing genes. However, even just a single base change out of our over three billion nucleotide genome can result in devastating and unfortunately rather common genetic diseases. 1 in 7,000 males worldwide are affected by muscular dystrophy, 1 in 12 people are carriers for Cystic Fibrosis, and 1 in every 365 African Americans has Sickle Cell Anemia. Each of these diseases can arise from a single nucleotide change.

In the last few years the CRISPR/Cas9 system has revolutionized what is possible in genome editing. However, the technology is not perfect due to its reliance on blunt end joining. There are often errors that happen when the DNA strands join back together. Because the normal CRISPR systems edits DNA, these errors are permanent. However, if we were to edit mRNA, then there is no risk of permanent damage in the genome.