Protein Modeling/CRISPR-Cas9 and Base Editing
The applications of CRISPR-Cas9 in base editing are the focus of Protein Modeling for the 2020 and 2021 seasons. This page details the pre-build protein APOBEC3A (PDB ID 5KEG) and the process of base editing - for more information on the CRISPR system in general, see Protein Modeling/CRISPR-Cas9.
APOBEC3A (A3A) is a member of the APOBEC superfamily of proteins. These proteins target cytidine nucleosides - instead of having the phosphate group which is present in a nucleotide, a nucleoside is simply a nitrogenous base bonded to a pentose sugar. Cytidine is a cytosine that is bonded to a ribose sugar. This nucleoside can be altered by the APOBEC enzyzmes through a process known as cytidine deamination, where an amino group is removed from the cytidine. This turns it into uridine - for that reason, this is known as C-to-U editing. This has the consequence of converting a C-G base pair into a U-A base pair (or T-A in DNA).
A3A is one of seven closely related APOBEC3 enzymes, all of which are located on chromosome 22. It has the highest catalytic activity among the APOBEC3 proteins. These enzymes are thought to be an important part of the immune system, restricting retroviruses like HIV.
A3A prominently features a five-stranded beta sheet, surrounded by six alpha helices. Because A3A is a single-domain enzyme (as opposed to other double-domain enzymes in the APOBEC3 group) there is an active zinc finger domain present as well. The substrate of this enzyme is ssDNA (single-stranded DNA) as opposed to dsDNA (double-stranded DNA), which allows for altering the DNA without inducing double-strand breaks.
The body's mechanisms of DNA repair have proved to be a stumbling block for scientists attempting to develop a mechanism for single-base editing. There are a variety of methods of DNA repair depending on the type of damage that took place - some damage can be directly reversed, while single strand breaks can use the remaining strand as a template to repair the issue. Double-stranded DNA breaks (DSBs) are the most hazardous to the cell, because they can result in instability or rearrangements.
There are two major pathways for double-stranded DNA repair: homology directed repair (HDR) and non-homologous end joining (NHEJ). HDR can be used when a homologous piece of DNA is present to use as a template for the repair - this typically takes place in the G2 or S phases after DNA has been replicated. NHEJ will occur if there is no homologous DNA to use for the repair. NHEJ simply reattaches the broken ends of the DNA instead of repairing them - this can result in random insertions or deletions (indels). However, NHEJ is much faster than HDR and it is used by the CRISPR-Cas9 system to repair the DSBs it induces. This may be useful if the goal is to knock-out a gene, but can cause problems in applications that require specific or precise single base editing.
Many genetic disorders are as a result of a point mutation, or an alteration of a single base. While the CRISPR-Cas9 system on its own is capable of editing entire genes, it is much less useful when attempting to edit a specific nucleotide. This is where the APOBEC cytidine deaminase enzymes are essential - they can edit a strand of DNA without creating a DSB. However, many of these enzymes only work on RNA or require ssDNA to function. CRISPR-Cas9 can be used to direct these cytidine deaminase enzymes towards specific areas of DNA that have been unpaired, allowing the enzyme to convert the cytosine base to a thymine.