Multi-nucleotide variants (MNVs) represent an important type of genetic variation and have biological and clinical significance. To simulate MNVs, we designed four dual-mutation base editors combining hA3A(Y130F), TadA8e(V106W), and protospacer adjacent motif (PAM)-flexible SpRY and selected cytosine and adenine base editor-SpRY (CABE-RY), which had the best editing performance, for further study. Characterization and comparison showed that CABE-RY had a smaller DNA editing window and lower RNA off-target edits than the corresponding single base editors. Thus, we have established a versatile tool to efficiently simulate MNVs over the genome, which could be very useful for functional studies on MNVs in humans.

Selective inhibition of targeted protein kinases is an effective therapeutic approach for treatment of human malignancies, which interferes phosphorylation of cellular substrates. However, a drug-imposed selection creates pressures for tumor cells to acquire chemoresistance-conferring mutations or activating alternative pathways, which can bypass the inhibitory effects of kinase inhibitors. Thus, identifying downstream phospho-substrates conferring drug resistance is of great importance for developing poly-pharmacological and targeted therapies. To identify functional phosphorylation sites involved in 5-fluorouracil (5-FU) resistance during its treatment of colorectal cancer cells, CRISPR-mediated cytosine base editor (CBE) and adenine base editor (ABE) are utilized for functional screens by mutating phosphorylated amino acids with two libraries specifically targeting 7779 and 10 149 phosphorylation sites. Among the top enriched gRNAs-induced gain-of-function mutants, the target genes are involved in cell cycle and post-translational covalent modifications. Moreover, several substrates of RSK2 and PAK4 kinases are discovered as main effectors in responding to 5-FU chemotherapy, and combinational treatment of colorectal cancer cells with 5-FU and RSK2 inhibitor or PAK4 inhibitor can largely inhibit cell growth and enhance cell apoptosis through a RSK2/TP53BP1/𝜸-H2AX phosphorylation signaling axis. It is proposed that this screen approach can be used for functional phosphoproteomics in chemotherapy of various human diseases.

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.