Design principles for Cas12a-mediated precision editing are defined and a scalable platform for genome-scale pooled variant engineering and phenotyping in yeast is established.
Abstract
Cas9 precision editing is increasingly predictable because guide, donor and target-context effects have been systematically characterized. Extending this framework to other nucleases is essential for installing variants outside convenient Cas9 target space. Cas12a provides a T-rich protospacer-adjacent motif (PAM) alternative, but determinants of efficient donor-templated Cas12a editing remain poorly defined. Here, we systematically dissected Cas12a precision editing in Saccharomyces cerevisiae across nuclease, direct repeat, expression, crRNA, donor, genomic context and time-course variables. Reporter and amplicon-sequencing assays showed that cleavage activity alone did not predict precise editing. Highly active configurations often reduced viability or lost edited alleles over time, whereas attenuated configurations better preserved programmed edits. Enhanced AsCas12a edited rapidly and tolerated shorter crRNAs, resulting in a narrower editing window, while an attenuated FnCas12a configuration edited more slowly but maintained higher viability and better distal-edit recovery. Alternative repair outcomes were rare, target-dependent, and further suppressed by LexA-FHA donor recruitment. To define design parameters at scale, we established a pooled Cas12a platform with 530 barcoded edit cassettes and recovered programmed edits for 70.2% of designs. Successful editing was reduced with TTTG PAMs, a C upstream of the PAM and at distal edit positions. Excluding these features increased the edited fraction to 85.4% and adding high predicted cleavage scores further elevated it to 91.4%. Applied retrospectively, these criteria also identified poorly edited loci in the targeted panels. Together, these data define design principles for Cas12a-mediated precision editing and establish a scalable platform for genome-scale pooled variant engineering and phenotyping in yeast.
Five previously uncharacterized MG102-like Cas9d orthologs are identified that share the hallmark genomic, sequence, and structural features of type II-D Cas9 and 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.
Qiaochu Wang, Ahmed Saleh, G. S. Rao et al.· bioRxiv· 0 citations
Base editors (BEs) enable efficient A-to-G or C-to-T conversions without double-stranded DNA cleavage, but their editing windows remain difficult to tune, limiting genome engineering flexibility. Here, we engineered CRISPR/Cas12b sgRNA by introducing MS2 hairpins to recruit an MS2-N55K-cytidine deaminase-UGI complex, enabling programmable control of the editing window. Three modified sgRNAs were generated by replacing two loop regions, each producing distinct editing hotspots in E. coli. The AID*Δ-MSBE system (sgRNA1.1) generated a window near the PAM with peak activity at C7-C9, while the CDA-MSBE system (sgRNA1.2) produced a distal window with peak activity at C20-C23. Both systems exhibited identical editing patterns in Bacillus subtilis. A dual-orthogonal system (MS2 and PP7) was constructed to simultaneously recruit two deaminase complexes, restoring the classic dCas12b CBE editing pattern. Rifampicin resistance assays confirmed high targeting specificity with low off-target effects. As proof of concept, the MSBEs were successfully employed for the flexible reprogramming of sfGFP fluorescence and the targeted evolution of the endogenous gene rpsE, respectively. Collectively, we developed the MSBEs with tunable editing hotspots, providing innovative tools to enhance the flexibility and accessibility of BEs for genome engineering.
A yeast selection platform is developed to engineer Cas9 with re-specified activity across multiple additional non-canonical PAMs in yeast, further demonstrating its utility as a general and programmable framework for expanding the therapeutic reach of precision genome editing.
Julia Tartaglia, Vivian Nguyen, John James Desmarais et al.· bioRxiv· 0 citations
Amongst the genome manipulation tools based on the versatile CRISPR-Cas system, prime editing is the most prominent one as a method that enables precise insertions, deletions, and substitutions without inducing double-strand breaks. Cas12a nucleases are widely used for genome editing and nucleic acid detection, owing to their unique properties; however, their relatively long PAM requirements limit their applicability.
We develop PAM-flexible Cas12a variants capable of functioning effectively within mammalian cells, thereby enabling the cleavage of targets previously inaccessible to Cas12a nucleases. Amongst the Lb-, As-, Mb-, and FnCas12a variants we develop, flexiAsCas12a (AsCas12a with flexible PAM recognition) is the most effective, expanding the range of recognized PAM sequences by Cas12a variants to include NATN, NCCN and GTCN sequences. Using the currently available Cas12a variants with relaxed PAM recognition (impLbCas12a, flexiAsCas12a, and enAsCas12a), we develop circular RNA-guided split prime editors and validate their functionality on non-canonical PAM sequences.
flexiAsCas12a joins the repertoire of Cas12a PAM variants, enabling access to an increasing number of target sequences by Cas12a nucleases.
CRISPR–Cas9 has revolutionised genome editing by enabling efficient and programmable modification of defined DNA sequences, with guide RNAs (gRNAs) serving as indispensable elements that direct Cas9 to specific genomic loci. Initially regarded as auxiliary components, gRNAs are now recognized as critical determinants of editing efficiency and specificity and have attracted growing attention as independent targets for engineering. Chemical modification, sequence optimisation, and structural alteration of gRNAs have been shown to enhance on‐target activity, suppress off‐target effects and cytotoxicity, and even achieve allele‐selective precision editing in a programmable manner. Moreover, advances in artificial intelligence and machine learning have markedly improved the predictive accuracy of gRNA design through large‐scale data analysis. Despite rapid progress, a consolidated review that integrates chemical, structural, and computational advances in gRNA engineering and highlights their translational potential for therapeutic genome editing has been lacking. This review uniquely addresses that gap by presenting an integrated framework that connects molecular design principles with clinical applicability.
Masaki Kawamata, S. Niwa, Atsushi Suzuki· Chemical Biology and Drug De...· 0 citations