CRISPR C-to-G Base Editors: A Therapeutic Promise
By Judy Ya-Hsuan Lin
CRISPR-guided DNA cytosine (C) and adenine (A) base editors are widely used for pyrimidine-to-pyrimidine and purine-to-purine transitions respectively. A recent Nature Biotechnology publication from J. Keith Joung’s laboratory at the Massachusetts General Hospital (MGH) report new CRISPR cytosine-to-guanine base editors (CGBEs) that induces pyrimidine to purine “transversion” alterations. The study demonstrates that CGBE1 is effective in editing cytosine (C) to guanine (G) while reducing levels of unwanted indel mutations in human cells.
A previous A-to-G editor ABEmax (ABE) could induce C-to-G edits at sites in which a cytosine was at position 6 of the protospacer, numbering starting from the position most distal to the protospacer adjacent motif (PAM). The team hypothesized that CGBE1 potentially induces such C-to-G edits effectively. They studied all possible modifications on several components within CGBE1, including an RNA-guided Cas9 nickase, an Escherichia coli-derived uracil DNA N-glycosylase (eUNG), and a rat APOBEC1 cytidine deaminase variant (R33A) which previously showed a reduction in off-target RNA and DNA editing activities.
Screening for the Best Construct of CGBE1
To optimize CGBE1’s effectiveness and performance in C-to-G base transversions in human cells, the research team tested several different combinations of modified components. From previous experiences, the team hypothesized that the heterodimeric TadA-based adenosine deaminase domain in ABEmax could deaminate C to uridine (U) at position 6 with subsequent base repair by cellular UNG at an abasic site, and thereafter the introduction of G took place, but its mechanism remained unknown.
The team also found that the addition of two uracil glycosylase inhibitor (UGI) domains to ABEmax could reduce C-to-G edits and indels in its previous research; they decided to remove the two UGIs from BE4max cytosine base editor (CBE) to create BE4max∆UGI construct to enhance more C-to-G edits than the wild-type BE4max. The team further modified the APOBEC1 part of BE4max∆UGI construct with R33A mutation and inserted an orthologous UNG from E. coli (eUNG) to increase C-to-G edits. The substitution to eUNG from the originally used human UNG (hUNG) was due to unchanged or decreasing numbers of edits. The construct that stood out among all other combinations was then finalized as eUNG-BE4max(R33A)∆UGI fusion, or CGBE1.
Comparison Between CGBE1 and Mini C-CGBE1
MiniCGBE1 is an eUNG-knockout form of CGBE1 that had slightly lower frequencies of editing (13% miniCGBE1 vs. 14.4%CGBE1) but lower indel frequencies than CGBE1 (8.5% miniCGBE1 vs. 10.4% CGBE1). Both CGBE1 and Mini C-CGBE1 showed that position 6, as well as extending from there, was the most effective editing spot for cytosines. Experimenting on two semi-alike editors might be providing an extra option for future researchers and saving costs for inserting an eUNG.
Fidelity & Efficacy Improvements
The team explored several ways to improve the fidelity and effectiveness of CGBE1 and miniCGBE1 across multiple human cancer cell lines—to the extent that medical personnel could possibly modify the cancer genome back to normal and generate healthy cells again. They found that CGBE1 and miniCGBE1 induced lower off-target DNA base edits than BE4ma solely after assessing their editing activities at 23 known SpCas9 off-target sites of five different guide RNAs (gRNAs). The team also broadened the targeting range of CGBEs during editing with the addition of SpCas9-NG and SpCas9-VRQR variants that recognized shorter NG and alternative NGA Pams.
To further ensure and substantiate their CGBEs a better choice, the team compared with new priming editing (PE) that introduced a diverse range of different edits, and the results demonstrated that CGBE owned higher frequencies of desired C-to-G edits than PE.
Future Directions & Applications
Although there was difficulty in identifying the reasons why the addition of the rat APOBEC1 R33A variant could increase the efficiency of C-to-G editing, the team discovered that C-to-G editing frequencies were dependent on cell type based on their four cell line results. In the future, they might look into the mechanistic parameters that might govern the frequencies and product purity of C-to-G edits.
The future application of CGBEs is anticipated to further expand base editing options for research and therapeutics. Unlike the present base editing techniques such as CBE and ABE, CGBEs could alter a pyrimidine to purine. If both CBE/ABE and CGBEs alter at the third nucleotide (codon) of the triplet, CGBEs gift 14 additional amino acid alterations that the current base editing cannot accomplish. Besides, CGBEs could show prominent effects on binding and gene expression by introducing transversion mutations into transcription factor binding sites, relative to transition mutations. Lastly, CGBEs could assist in correcting disease-causing mutations. With these applications, many envision further progress with more sophisticated C-to-G editors.
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