Gene therapy would benefit from a miniature CRISPR system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in eukaryotic cells. One of the most compact CRISPR-associated nucleases yet discovered is the archaeal Un1Cas12f1. However, Un1Cas12f1 and its variants have very low activity in eukaryotic cells. In the present study, we redesigned the natural guide RNA of Un1Cas12f1 at five sites: the 5′ terminus of the trans-activating CRISPR RNA (tracrRNA), the tracrRNA–crRNA complementary region, a penta(uridinylate) sequence, the 3′ terminus of the crRNA and a disordered stem 2 region in the tracrRNA. These optimizations synergistically increased the average indel frequency by 867-fold. The optimized Un1Cas12f1 system enabled efficient, specific genome editing in human cells when delivered by plasmid vectors, PCR amplicons and AAV. As Un1Cas12f1 cleaves outside the protospacer, it can be used to create large deletions efficiently. The engineered Un1Cas12f1 system showed efficiency comparable to that of SpCas9 and specificity similar to that of AsCas12a.

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...

As a member of the epidermal growth factor receptor (EGFR) family, ERBB3 plays an essential role in development and disease independent of inherently inactive kinase domain. Recently, ERBB3 has been found to bind to ATP and has catalytic activity in vitro. However, the biological function of ERBB3 kinase activity remains elusive in vivo. Here we have identified the physiological function of inactivated ERBB3 kinase activity by creating Erbb3‐K740M knockin mice in which ATP cannot bind to ERBB3. Unlike Erbb3 knockout mice, kinase‐inactive Erbb3K740M homozygous mice were born in Mendelian ratios and showed normal development. After dextran sulfate sodium‐induced colitis, the kinase‐inactive Erbb3 mutant mice showed normal recovery. However, the outgrowth of ileal organoids by neuregulin‐1 treatment was more attenuated in Erbb3 mutant mice than in WT mice. Moreover, in combination with the ApcMin mouse, the proportion of polyps less than 1 mm in diameter in mutant mice was higher than in control mice and an increase in the number of apoptotic cells was observed in polyps from mutant mice compared with polyps from control mice. Taken together, the ERBB3 kinase activity contributes to the outgrowth of ileal organoids and intestinal tumorigenesis, and the development of ERBB3 kinase inhibitors, including epidermal growth factor receptor family members, can be a potential way to target colorectal cancer.

Genetically engineered mouse models through gene deletion are useful tools for analyzing gene function. To delete a gene in a certain tissue temporally, tissue-specific and tamoxifen-inducible Cre transgenic mice are generally used. Here, we generated transgenic mouse with cardiac-specific expression of Cre recombinase fused to a mutant estrogen ligand-binding domain (ERT2) on both N-terminal and C-terminal under the regulatory region of human vasoactive intestinal peptide receptor 2 (VIPR2) intron and Hsp68 promoter (VIPR2-ERT2CreERT2). In VIPR2-ERT2CreERT2 transgenic mice, mRNA for Cre gene was highly expressed in the heart. To further reveal heart-specific Cre expression, VIPR2-ERT2CreERT2 mice mated with ROSA26-lacZ reporter mice were examined by X-gal staining. Results of X-gal staining revealed that Cre-dependent recombination occurred only in the heart after treatment with tamoxifen. Taken together, these results demonstrate that VIPR2-ERT2CreERT2 transgenic mouse is a useful model to unveil a specific gene function in the heart.