Hypercompact adenine base editors based on a Cas12f variant guided by engineered RNA

Abstract

Transposon-associated transposase B (TnpB) is deemed an ancestral protein for type V, Cas12 family members, and the closest ancestor to UnCas12f1 due to its high sequence similarity. Previously, we reported a set of engineered guide RNAs supporting high indel e ciency for Cas12f1 in human cells. Here, we suggest a new technology whereby the engineered gRNAs also manifest highly e ciency programmable endonuclease activity for TnpB, termed TaRGET (TnpB-augment RNA-based Genome Editing Technology). Having this feature in mind, we established TnpB-based adenine base editors. A codon-optimized Tad-Tad mutant (V106W, D108Q) dimer fused to the C-terminus of dTnpB (D354A) showed the highest levels of A-to-G conversion. The limited targetable sites for TaRGET-ABE were expanded by either developing several PAM variants of TnpB or by engineering TnpB and optimizing deaminases at PAM-distal and PAM-proximal sites, respectively. When delivered by AAV, the TaRGET-ABE showed potent A-to-G conversion rates in human cells. Collectively, the TaRGET-ABE will contribute to improving precise genome-editing tools that can be delivered by AAV, thereby harnessing the development of CRISPR-based gene therapy. MainLiving organisms are subjected to spontaneous genetic variations, which form the basis for biodiversity and evolution. The random nature of genetic variation as well as non-biological factors are also responsible for a variety of genetic disorders. Of the types of genetic variations identi ed in humans, single-nucleotide variations (SNVs) account for nearly half of disease-related mutations 1 . This suggests that the development of site-speci c, precise genome editing tools holds promise for the treatment of otherwise intractable genetic disorders. CRISPR/Cas-mediated base-editing systems have addressed the unsatisfactory modi cation e ciency of homology-directed repair (HDR) by exploiting the high genetargeting capability of the CRISPR/Cas systems. The catalytically inactive Cas (dCas) or nickase Cas (nCas) fused to naturally occurring or engineered deaminases led to highly e cient single-nucleotide alterations including the C:G-to-T:A 2 and A:T-to-G:C 3 conversion. The introduction of an E. coli-derived uracil DNA N-glycosylase also enabled C:G-to-G:C transversion 4 . Moreover, the base editing systems guarantee higher levels of safety from a clinical view point because they enable precise genome editing with negligible or low levels of double-strand breaks (DSBs). In addition to such innate high e ciency and safety, base editing systems have further evolved in various aspects including decreased off-target editing, enhanced conversion speci city, broadened editing windows, increased editing e ciency, and control of unwanted editing [5][6][7] .Despite the dramatic improvements of base-editing system per se, the challenges on the delivery side remain a major hurdle preventing base editors from being widely used in clinical applications 8 . Adenoassociated viruses (AAVs) are considered as a validated delivery platform due to the...

TadA reprogramming to generate potent miniature base editors with high precision

“…Four representative d12fABEs-8e (106 W) were further evaluated across 12 endogenous sites by targeted amplicon sequencing. It was shown that N-d12fABE-8e (106 W) displayed an editing window at A2-A4 (an average of 9.5%~11.9% editing frequency, R of PAM TTTR counted as 0) distal to PAM, similar to reported miniABEs 7,11 (Fig. 1c).…”

“…Though split base editors have been generated to achieve efficient delivery by dual AVVs 41 , miniature base editors are urgently needed to enable all-in-one AAV delivery in vivo for therapeutic applications. Actually, previous studies have only generated functional miniABEs with Cas12f or its orthologue systems 7,11 , and no functional miniCBEs have been reported previously. Here we generate a series of potent miniABEs and miniCBEs with diversified editing signatures composed of various engineered deaminases and hypercompact CRISPR-Cas12f system, with the help of flow cytometry (Supplementary Fig.…”

“…Hypercompact CRISPR-Cas12f systems have been engineered for functional miniABEs [6][7][8][9][10] . Nevertheless, Cas12f-derived miniABEs are still hindered by modest A-to-G editing activity and restricted editing scopes 7,11 . Moreover, no functional miniCBEs have been reported so far.Classical ABEs contained evolved tRNA-specific adenosine deaminases from Escherichia coli (ecTadA) (evolved ecTadA variant, simplified as TadA*) while CBEs contained cytidine deaminases 2,3,12 .…”

“…Hypercompact CRISPR-Cas12f systems have been engineered for functional miniABEs [6][7][8][9][10] . Nevertheless, Cas12f-derived miniABEs are still hindered by modest A-to-G editing activity and restricted editing scopes 7,11 . Moreover, no functional miniCBEs have been reported so far.…”

Recent Advances in CRISPR-Cas Technologies for Synthetic Biology

“…Deactivated CasMINI-mediated adenine base editor (dCasMINI-ABE) showed the most efficient A to G conversion in a narrow window (3–4 bp downstream region from the PAM). TnpB-based ABE, made through the fusion of the C-terminus of dTnpB with a modified dimer of TadA adenosine deaminase, facilitates A to G conversion in the PAM-proximal region (Kim et al, 2022 ). The conversion efficiency of TnpB-based ABE was higher than that of Cas12f-based ABE, but still lower than that of SpCas9-based ABE.…”