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New CRISPR Base Editors Induce Targeted DNA Transversions


NEW YORK – Two research teams have developed new CRISPR base editors that induce targeted DNA transversions, changing C to G in human cells and changing C to A in bacteria. The new technology could be developed into therapies for diseases that are caused by DNA transversion point mutations, and could also be used to elucidate the molecular underpinnings of those conditions. 


Until now, base editors have been able to correct base transitions: cytosine base editors (CBEs) convert C-G base pairs into T-A base pairs at targeted locations in the genome, and adenine base editors (ABEsconvert A-base pairs into G-C base pairs. The biology of DNA — cytosine and thymine being pyrimidines and adenine and guanine being purines — has made transversions much more difficult to accomplish in a targeted manner. 


In Nature Biotechnology on MondayMassachusetts General Hospital researchers led by Julian Grünewald and J. Keith Joung described their development of two base editor architectures that can efficiently induce targeted C-to-G base transversions, with reduced levels of unwanted C-to-A or C-to-T changes and indel mutations. One of these C-to-G base editors — called CGBE1 — is composed of an RNA-guided Cas9 nickase, an Escherichia coli-derived uracil DNA N-glycosylase (eUNG), and a rat APOBEC1 cytidine deaminase variant (R33A) previously shown to have reduced off-target RNA and DNA editing activities, the researchers said. They noted that CGBE1 can efficiently induce C-to-G edits, particularly in AT-rich sequence contexts in human cells.  


They also developed a smaller version of the editor called miniCGBE1 by removing the eUNG domain, thereby reducing indel frequencies and only modestly decreasing editing efficiency. CGBE1 and miniCGBE1 enable C-to-G edits and could serve as a basis for optimizing C-to-G base editors for research and therapeutic applications, the researchers added. 


"C-to-G base editors can target and correct mutations that cannot be targeted with either ABEs or CBEs," Grünewald said. He noted that it's not entirely possible to quantify the number of diseases that could be corrected with CGBEs because many known mutations are only disease-associated rather than disease-causing — some estimates have put the number of G/C SNPs associated with human disease at approximately 3,000.  


"Around 11 percent of known disease-associated or pathogenic single nucleotide variants could in theory be reversed by CGBEs," Grünewald added"At this stage, it is too early to envision whether CGBEs will ultimately be applied as therapeutics as there is much further optimization and understanding of their activities that will be required. But we do note that there are numerous research applications enabled by CGBEs that we envision can, in turn, be used to better understand various diseases."  


The researchers' efforts to develop a C-to-G base editor began when they observed that the A-to-G editor ABEmax could induce unexpected C-to-G edits at sites in which a C was present at position six of the protospacer. Given this observation, they hypothesized that they could induce these edits more efficiently by modifying the BE4max CBE, which harbors the rat APOBEC1 cytidine deaminase — which is intended to deaminate cytosines. When they removed the two UGIs from BE4max to create BE4max∆UGI, they saw an increase in C-to-G edits relative to wild-type BE4max in HEK293T cells.  


After testing a variety of architectures, the investigators focused on the APOBEC1 R33A mutation, which they had previously used to decrease off-target RNA editing while substantially preserving the efficiency and increasing the precision of on-target DNA editing by CBEs. They found that adding R33A into the BE4max∆UGI with a human UNG (hUNG) increased C-to-G editing frequencies with three of the seven guide RNAs tested in HEK293T cells, while leaving editing frequencies essentially unaltered with the other fourThey eventually replaced the hUNG with the eUNG to increase the efficiency of C-to-G edits.  


To more comprehensively characterize the resulting CGBE1 editor, the researchers tested its activity with 18 additional gRNAs in human HEK293T cells. They observed highly efficient C-to-G edits for four of the 18 sites, with mean editing frequencies ranging from 41.7 percent to 71.5 percent and only very low levels of C-to-T or C-to-A byproducts. When these data were combined with the results obtained from the initial seven gRNAs, CGBE1 induced C-to-G editing with mean frequencies of 20 percent or higher at 14 of the 25 sites tested, they noted.  


Side-by-side comparisons of miniCGBE1 with CGBE1 at the same 25 sites the researchers had previously tested showed that the frequencies of editing observed with miniCGBE1 were comparable but moderately lower at six out of 25 sites tested, whereas the indel frequencies induced by miniCGBE1 were lower at 15 out of 25 sites.  


Further, the researchers said, when they assessed gRNA-dependent DNA off-target editing, they found that CGBE1 and miniCGBE1 induced fewer off-target DNA base edits than BE4max, that CGBE-induced indels could occur at off-target sites, and that indels were reduced with miniCGBE1 relative to CGBE1.  


The question of off-target edits could be considered an especially important one to ask when analyzing the advantages or disadvantages of newly developed base editing technology. In mid-2019, several studies were published showing that base editors could induce tens of thousands of off-target mutations in RNA, induce SNVs with frequencies more than 20-fold higher than the spontaneous mutation rate, or could cause extensive transcriptome-wide off-target RNA editing in human cells.  


These findings led to extensive engineering of new ABE and CBE variants to correct those problems, including SECURE-CBE and SECURE-ABE variants developed by Joung, Grünewald, and their colleagues, which they devised in order to reduce off-target RNA-editing activity. 


"The benefit of our CGBE is that it's modeled after the SECURE-CBE variant that we published last year and that showed greatly reduced off-target RNA editing," Grünewald said"This variant was also shown to exhibit reduced DNA off-target effects. We also show in our current work that gRNA-dependent off-target base editing with our CGBEs is lower than with some CBEs. Of course, we are continuing to work on ways to further reduce unwanted off-target effects of these and other base editors." 


Transversions in Bacteria  


In a second paper published on Monday in Nature Biotechnologyresearchers from the Chinese Academy of Sciences, the University of Edinburgh, and Tianjin Normal University also described their development of a base editor that can induce C-to-G transversions in mammalian cells, and further said they engineered an editor that can induce C-to-A transversions in E. coli.  


The glycosylase base editors (GBEs) consist of a Cas9 nickase, a cytidine deaminase, and a uracil-DNA glycosylase (Ung), the researchers said. Ung excises the U base created by the deaminase, forming an apurinic/apyrimidinic (AP) site that initiates the DNA repair process. In E. coli, they used activation-induced cytidine deaminase (AID) to construct AID-nCas9-Ung and found that it converted C to A with an average specificity of 93.8 percentand efficiency of 87.2 percent. 


For mammalian cells, the investigators replaced AID with rat APOBEC1 (APOBEC-nCas9-Ung). When they tested APOBEC-nCas9-Ung at 30 endogenous sites, they observed C-to-G conversions with a high editing specificity at the sixth position of the protospacer between 29.7 percent and 92.2 percent, and an editing efficiency between 5.3 percent and 53 percent. The APOBEC-nCas9-Ung supplements current ABEs and CBEs, and could be used to target G/C disease-causing mutations, they added. 


The researchers also analyzed their data to see whether Ung-nCas9-AID had off-target effects in E. coliand found that no notable off-target events occurred at any of the 10 loci they sequenced. In the mammalian cells, the mutation rates in off-target sites by APOBEC-nCas9-Ung ranged from 2 percent to 3 percentThey also found that the average editing efficiency of C-to-A conversions by the GBE was 2.7 percent, suggesting that GBEs could be applied in scenarios in which one C needs to be edited in the context of other C bases.  


According to Grünewald, there are several directions that CRISPR base transversion technology could now be taken. For example, in June, he was first author on a paper published in Nature Biotechnology describing a dual-deaminase CRISPR base editing platform that combined the abilities of ABEs and CBEs to allow for concurrent A-to-G and C-to-T edits. The potential of adding a CGBE to that dual deaminase platform is an idea "worth exploring," he said. 


Grünewald also noted that the development of a C-to-A editor in mammalian cells could have useful applications in both medical and biotechnological scenarios. 


"The next logical steps would be the development of more efficient CGBEs, as well as editors that unlock the remaining transversionsIn principle, it would be desirable to have an entire suite of different base editors to specifically introduce any given type of single base substitution," he said, adding that some research teams may also want to focus on improving the efficiency of delivering base editors to cells and tissues in vivo.