Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo

Abstract

CRISPR/Cas9-based base editing tools enable precise genomic installation and hold great promise for gene therapy, whereas the big size of Cas9 nucleases and its reliability on specific protospacer adjacent motif (PAM) sequences as well as target site preferences restrict the extensive applications of base editing tools. Here, we generate two cytosine base editors (CBEs) by fusing cytidine deaminases with two compact codon-optimized Cas9 orthologs from Streptococcus_gordonii_str._Challis_substr._CH1 (ancSgo-BE4) and Streptococcus_thermophilus_LMG_18311 (ancSth1a-BE4), which are much smaller than Streptococcus pyogenes (SpCas9) and recognize NNAAAG and NHGYRAA PAM sequences, respectively. Both CBEs display high activity, high fidelity, a different editing window, and low by-products for cytosine base editing with minimal DNA and RNA off-targeting activities in mammalian cells. Moreover, both editors show comparable or higher editing efficiencies than two engineered SpCas9 variant (SpCas9-NG and SpRY)-based CBEs in our tested target sites, which perfectly match the PAM sequences for ancSgo-BE4 or ancSth1a-BE4. In addition, we successfully generate two mouse models harboring clinically relevant mutations at the Ar gene via ancSgo-BE4 and ancSth1a-BE4, which display androgen insensitivity syndrome and/or developmental lethality in founder mice. Thus, the two novel CBEs broaden the base editing tool kits with expanded targeting scope and window for efficient gene modification and applications, respectively.

Cas9‐orthologue‐mediated cytosine and adenine base editors recognizing NNAAAA PAM sequences

“…Indeed, the replacement of APOBEC1 with APOBEC3A (Y130F) significantly improved the performance of SsiCas9-mediated CBE in GC and non-GC targets (Figure 2). Combining with our recently reported findings, [48] these results indicated that engineering CBEs with APOBEC3A might be a universal strategy to improve the sequence-context bias. However, considering the relatively high indel rates induced by ancSsi-A3A-BE4, when sufficient targeting efficiency can be achieved using ancSsi-BE4, ancSsi-A3A-BE4 can serve as a strong backup tool for inducing targeted cytosine conversion.…”

“…To broaden the usage of base editing with various PAM recognition capabilities, we selected SsiCas9, which is smaller than SpCas9 (1121 vs. 1368 residues, Figure S1A), has high cleavage activity at 37°C, and recognizes NNAAAA PAM sequence in in vitro biochemical assays (Figure S1B), as a potentially novel Cas9 protein from 79 Cas9 homologs library [ 34 ] for base editor engineering. Compared to the scaffold sequence of the gRNAs for SpCas9, 45.56% of the nucleotides were conserved with those of SsiCas9 (Figure S1C).…”

“…Thus, we propose that SsiCas9 has the potential to be engineered with less restrictive PAM requirements, similar to Nme2Cas9. [47] Considering that the off-targeting activity of the CRISPR/Cas9 system may largely impede basic research and restrict practical deployment, [48] we examined the off-targeting activity of ancSsi-BE4 when targeting three endogenous loci (Ssi-1, Ssi-4, and Ssi-7) in HEK293T cells. Potential off-target sites with ≤5 mismatched nucleotides were predicted using the online tool Cas-OFFinder3, and the genomic regions containing these off-target sites were amplified by PCR and subjected to targeted deep sequencing.…”

“…Recently, we reported two compact Cas9 orthologue‐based CBEs, ancSgo‐BE4 and ancSth1a‐BE4, that can recognize NNAAAG and NHGYRAA PAM sequences, respectively, with high efficiency and accuracy. [ 34 ] In the present study, we developed a novel CBE or ABE by fusing a Cas9 ortholog from Streptococcus sinensis (named as SsiCas9) within BE4max, ABEmax, or ABE8e, which can achieve highly efficient base conversion with low off‐targeting activity and indel rates in mammalian cells and early mouse embryos. It broadens the toolbox complexity for base editing and further validates the versatility of our strategy for genome editing.…”

Genome Editing for Sustainable Crop Improvement and Mitigation of Biotic and Abiotic Stresses

“…Base editing (BE) is a technique that directly converts one target genomic DNA base into another at a targeted locus without producing a double-stranded break. Combining cytosine or adenine deaminases with CRISPR-Cas9, a range of cytosine base editors (CBEs) and adenine base editors (ABEs) has been developed in recent years [ 66 , 67 , 68 ]. These varieties allow exact C-to-T or A-to-G base conversions without causing a DSB.…”