Biology subjects

Kazlak, A. M.

Publications and source records attributed to Kazlak, A. M..

3 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↗

bioRxiv2025

Characterization and engineering of highly efficient Cas12j genome editors

The large size of CRISPR-Cas enzymes limits their delivery for therapeutic applications. Cas12j nucleases offers hypercompact alternative but show moderate editing efficiency. To overcome this limitation, we identified eight novel Cas12j orthologues (Cas12j-11 to Cas12j-18) from viral metagenomes. All showed low editing activity in mammalian cells. We engineered T5 exonuclease-Cas12j fusions (T5Exo-Cas12j), two of which, T5Exo-Cas12j-12, and -18 exhibited up to 42% editing in HEK293T and 9% in K-562 cells, outperforming wild-type Cas12j counterparts and comparable to LbCas12a. Intriguingly, robust in cellula editing in both HEK293T and K-562 cells was strictly dependent on the presence of 5'-TAC trinucleotides within the target DNA sequence. Furthermore, we fused the Cas12j orthologues with the TadA8e deaminase and developed base editors, termed Be-(d)Cas12j. Among these, Be-(d)Cas12j-13 demonstrated efficient A-to-G base conversion in mammalian cells. This study expands the CRISPR toolbox by characterizing and engineering novel Cas12j orthologues into compact, high-efficiency genome editors.

bioengineering↗