Explore the vast range of titles available.

Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, “AsCas12a Ultra”, that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines. The utility of AsCas12a can be limited to poor editing efficiency. Here the authors identify a variant, “AsCas12a Ultra”, that has high on-target specificity demonstrated through editing of clinically relevant T cell genes.

The ability to precisely modify RNA offers opportunities to manipulate the flow of genetic information and influence transcript stability, localization and translation. RNA-targeting technologies enable RNA knockdown, base editing and -splicing, but more extensive transcript changes typically require genome editing or rely on the endogenous splicing machinery. Based on the ability of type III-A CRISPR-Csm complexes to catalyze programmable RNA cleavage in human cells, we investigated their potential to induce site-specific deletions while leaving the remainder of the transcript intact. Our data show that CRISPR-Csm complexes can generate short and long RNA excisions within a target transcript, and that the efficiency of this process is enhanced by fusion of Csm to the RNA ligase RtcB. Furthermore, cleavage of two different transcripts can trigger subsequent -ligation of the cleaved products into a chimeric transcript (\"spligation\"). Finally, we apply spligation to endogenous transcripts, using Csm to generate recombinant mRNA in cells independent of canonical splice sites. Collectively, this approach enables new forms of precise RNA manipulation in cells with potential applications in human disease.

Genome editing enzymes have vast therapeutic potential. However, achieving sufficient delivery remains a major challenge, because editing machinery is confined to the subset of transfectable cells in a tissue. Here, we tested the possibility that genome editing could be amplified by programming transfected cells to produce and transfer editing enzymes in lipid vesicles to neighboring cells. Our data show that this NANoparticle-Induced Transfer of Enzyme (NANITE) strategy tripled editing efficiency in cultured cells relative to non-spreading controls. Furthermore, a single intravenous injection of the NANITE plasmid into mice induced ~3-fold higher levels of liver editing at the locus relative to non-spreading controls, with corresponding reductions in serum transthyretin levels. Amplifying therapeutic enzymes offers a nonviral and non-infectious strategy to overcome low delivery efficiencies and reduce effective dose requirements.

CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture and human therapeutics. However, their dependence on a 5'-TTTV-3' protospacer-adjacent motif (PAM) next to DNA target sequences restricts Cas12a's gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Cas12a (LbCas12a) with expanded PAM recognition. The resulting Cas12a variants use a range of non-canonical PAMs while retaining recognition of the canonical 5'-TTTV-3' PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5'-NYHV-3' PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.

RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a 20-base-pair DNA sequence adjacent to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical and cell-based assays on Cas9 ( Cas9) variants with wide-ranging genome editing efficiencies that differ in PAM binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.

The recruitment and retention of physicians in rural communities is a challenge throughout Canada and across the globe. In 1976, a group of medical students profiled rural communities with medical practices and produced a summary report entitled Medical Practice in Saskatchewan. Our objective was to repeat the 1976 study and to identify factors that motivate physicians to select rural locations for practice. Physicians practising in rural Saskatchewan were interviewed in 2011 and 2012. Through qualitative, inductive analysis, we identified themes that drove the recruitment and retention of physicians. Sixty-two physicians were interviewed and 105 communities profiled. Of the physicians interviewed, 21 noted that the ability to practise full-scope family medicine and having the freedom to practise as they desire was important for recruitment, and 43 reported that these factors influenced their decision to remain in a community. Attraction to a rural lifestyle (cited by 17 physicians), having a rural background (13) and having ties to a specific community (12) were important for recruitment. Feeling appreciated by patients (45), one's spouse and/or family enjoying the community (41), and integration into the community (38) were important factors for retention. The decision to practise in a rural location correlates with a desire for a broad and varied scope of practice, being attracted to a rural lifestyle and having rural roots. Once physicians establish a rural practice, they are more likely to stay if they can continue a broad scope of practice, if they feel appreciated by their patients, and if their spouses and family are happy in the community.

TnpB is a compact RNA-guided endonuclease and evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of TnpB (Ymu1 TnpB). DNA unwinding proceeds through formation of a partially unwound intermediate state to a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, Ymu1-WFR, stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage and increased genome editing . These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.

The Ku complex performs multiple functions inside eukaryotic cells, including protection of chromosomal DNA ends from degradation and fusion events, recruitment of telomerase, and repair of double-strand breaks (DSBs). Inactivation of Ku complex genes YKU70 or YKU80 in cells of the yeast Saccharomyces cerevisiae gives rise to mutants that exhibit shortened telomeres and temperature-sensitive growth. In this study, we have investigated the mechanism by which overexpression of telomerase suppresses the temperature sensitivity of yku mutants. Viability of yku cells was restored by overexpression of the Est2 reverse transcriptase and TLC1 RNA template subunits of telomerase, but not the Est1 or Est3 proteins. Overexpression of other telomerase- and telomere-associated proteins (Cdc13, Stn1, Ten1, Rif1, Rif2, Sir3, and Sir4) did not suppress the growth defects of yku70 cells. Mechanistic features of suppression were assessed using several TLC1 RNA deletion derivatives and Est2 enzyme mutants. Supraphysiological levels of three catalytically inactive reverse transcriptase mutants (Est2-D530A, Est2-D670A, and Est2-D671A) suppressed the loss of viability as efficiently as the wild-type Est2 protein, without inducing cell senescence. Roles of proteins regulating telomere length were also determined. The results support a model in which chromosomes in yku mutants are stabilized via a replication-independent mechanism involving structural reinforcement of protective telomere cap structures.

A key barrier to translation of biomedical research discoveries is a lack of understanding among scientists regarding the complexity and process of implementation. To address this challenge, the National Science Foundation's Innovation Corps™ (I-Corps™) program trains researchers in entrepreneurship. We report results from the implementation of an I-Corps™ training program aimed at biomedical scientists from institutions funded by the National Center for Advancing Translational Sciences (NCATS). National/regional instructors delivered 5-week I-Corps@NCATS short courses to 62 teams (150 individuals) across six institutions. Content included customer discovery, value proposition, and validating needs. Teams interviewed real-life customers and presented the value of innovations for specific end-users weekly, culminating in a \"Finale\" featuring their refined business thesis and business model canvas. Methodology was developed to evaluate the newly adapted program. National mixed-methods evaluation assessed program implementation, reach, effectiveness using observations of training delivery and surveys at Finale ( = 55 teams), and 3-12 months post-training ( = 34 teams). Innovations related to medical devices (33%), drugs/biologics (20%), software applications (16%), and diagnostics (8%). An average of 24 interviews was conducted. Teams reported increased readiness for commercialization over time (83%, 9 months; 14%, 3 months). Thirty-nine percent met with institutional technology transfer to pursue licensing/patents and 24% pursued venture capital/investor funding following the short courses. I-Corps@NCATS training provided the NCATS teams a rigorous and repeatable process to aid development of a business model based on customer needs. Outcomes of this pilot program support the expansion of I-Corps™ training to biomedical scientists for accelerating research translation.