Biology subjects

Aman, R.

Publications and source records attributed to Aman, R..

4 recordsLinked to original sources

bioRxiv2026

Compact type II-D Cas9 nucleases for efficient and specific genome editing

Compact CRISPR nucleases are attractive for therapeutic genome editing because their small coding sequences facilitate delivery by adeno-associated virus. Type II-D Cas9 (Cas9d) enzymes constitute the most compact Cas9 subtype, yet only a few orthologs have demonstrated mammalian genome-editing activity, leaving it unclear whether this activity is general or exceptional. Here, we mined the IMG/M metagenomic database and identified five previously uncharacterized MG102-like Cas9d orthologs ([~]950 amino acids) that share the hallmark genomic, sequence, and structural features of type II-D Cas9. Two of them, Cas9d-1 and Cas9d-4, recognized a 5-NRC-3 protospacer-adjacent motif and edited endogenous human loci with efficiencies up to 20.1%, exceeding Streptococcus pyogenes Cas9 at one site, while producing deletion-biased outcomes and no detectable off-target activity. Notably, both orthologs edited more efficiently than the sole previously validated member of this lineage, MG102-2, when assayed side by side under identical conditions. These findings establish compact MG102-like Cas9d orthologs as robust and specific genome editors and provide promising, single-AAV- compatible scaffolds for in vivo therapeutic genome editing.

bioengineering↗

bioRxiv2026

Compact type II-C Cas9 nucleases with expanded PAM access and high fidelity for therapeutic genome editing

Compact type II-C Cas9 nucleases are attractive for therapeutic genome editing because their small size enables packaging into adeno-associated viral (AAV) vectors, and their extended protospacer-adjacent motifs (PAMs) reduce off-target cleavage while expanding targeting scope. Yet characterized type II-C orthologs have edited mammalian cells far less efficiently than the canonical SpCas9. Here, we used embedding-based metagenomic mining of >4.7 x 10 proteins, combined with AlphaFold3 structure prediction and locus-context analysis, to identify three previously uncharacterized compact type II-C Cas9 orthologs, NsuCas9 (1,092 aa), PsuCas9 (1,084 aa), and GfoCas9 (1,074 aa), and benchmarked them in vitro and in human HEK293T cells. All three are robust RNA-guided nucleases with distinct PAM specificities (N CC, N NYAA, and N RHAA, respectively), divergent thermal profiles, and asymmetric sgRNA cross-compatibility. In human cells, PsuCas9 with an N ATAA PAM reaches 78.4% indels and matches or exceeds SpCas9 at multiple loci, representing the first natural compact type II-C ortholog reported to do so, while GfoCas9 and NsuCas9 add complementary coverage. All three show a strong deletion-biased repair signature and no detectable editing across 33 predicted off-target sites. These compact, high-fidelity nucleases expand the CRISPR targeting space for AAV-deliverable therapeutic editing.

bioengineering↗

bioRxiv2021

A Calcium/Palmitoylation Switch Interfaces the Signaling Networks of Stress Response and Transition to Flowering

The precise timing of flowering in adverse environments is critical for plants to secure reproductive success. We report a novel mechanism controlling the time of flowering by which the palmitoylation-dependent nuclear import of protein SOS3/CBL4, a Ca2+-signaling intermediary in the plant response to salinity, results in the selective stabilization of the flowering time regulator GIGANTEA inside the nucleus under salt stress, while degradation of GIGANTEA in the cytosol releases the protein kinase SOS2 to achieve salt tolerance. S-acylation of SOS3 was critical for its nuclear localization and the promotion of flowering, but dispensable for salt tolerance. SOS3 interacted with the photoperiodic flowering components GIGANTEA and FKF1 on the CONSTANS gene promoter to sustain the transcription of CO and FT under salinity. Thus, SOS3 acts as a Ca2+- and palmitoylation-dependent molecular switch that fine-tunes flowering in a saline environment through the shared spatial separation and selective stabilization of GIGANTEA. The SOS3 protein connects two signaling networks to co-regulate stress adaptation and time of flowering. Short summaryS-acylation promoted the nuclear import of SOS3/CBL4 for the selective stabilization of the photoperiodic floral regulator GIGANTEA to fine-tune flowering time in a saline environment. Spatial separation of SOS3 acts as a molecular switch co-regulating stress adaptation and time of flowering.

plant biology↗

bioRxiv2017

RNA virus interference via CRISPR/Cas13a system in plants

CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants. CRISPR/Cas13a produced interference against green fluorescent protein (GFP) expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. crRNAs targeting the HC-Pro and GFP sequences exhibited better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs. Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses, and for other RNA manipulations in plants.

molecular biology↗