Biology subjects

Panwala, R.

Publications and source records attributed to Panwala, R..

3 recordsLinked to original sources

bioRxiv2024

Rational Design of Enhanced Nme2Cas9 and Nme2SmuCas9 Nucleases and Base Editors

CRISPR-Cas genome editing tools enable precise, RNA-guided modification of genomes within living cells. The most clinically advanced genome editors are Cas9 nucleases, but many nuclease technologies provide only limited control over genome editing outcomes. Adenine base editors (ABEs) and cytosine base editors (CBEs) enable precise and efficient nucleotide conversions of A:T-to-G:C and C:G-to-T:A base pairs, respectively. Therapeutic use of base editors (BEs) provides an avenue to correct approximately 30% of human pathogenic variants. Nonetheless, factors such as protospacer adjacent motif (PAM) availability, accuracy, product purity, and delivery limit the full therapeutic potential of BEs. We previously developed Nme2Cas9 and its BE derivatives, including ABEs compatible with single adeno-associated virus (AAV) vector delivery, in part to enable editing near N4CC PAMs. Further engineering yielded domain-inlaid BEs with enhanced activity, as well as Nme2Cas9/SmuCas9 chimeras that target single-cytidine (N4C) PAMs. Here we further enhance Nme2Cas9 and Nme2SmuCas9 editing effectors for improved efficiency and vector compatibility through site-directed mutagenesis and deaminase linker optimization. Finally, we define the editing and specificity profiles of the resulting variants by using paired guide-target libraries.

molecular biology↗

bioRxiv2023

Interface-guided phenotyping of coding variants in the transcription factor RUNX1 with SEUSS

Understanding the consequences of single amino acid substitutions in cancer driver genes remains an unmet need. Perturb-seq provides a tool to investigate the effects of individual mutations on cellular programs. Here we deploy SEUSS, a Perturb-seq like approach, to generate and assay mutations at physical interfaces of the RUNX1 Runt domain. We measured the impact of 115 mutations on RNA profiles in single myelogenous leukemia cells and used the profiles to categorize mutations into three functionally distinct groups: wild-type (WT)-like, loss-of-function (LOF)-like and hypomorphic. Notably, the largest concentration of functional mutations (non-WT-like) clustered at the DNA binding site and contained many of the more frequently observed mutations in human cancers. Hypomorphic variants shared characteristics with loss of function variants but had gene expression profiles indicative of response to neural growth factor and cytokine recruitment of neutrophils. Additionally, DNA accessibility changes upon perturbations were enriched for RUNX1 binding motifs, particularly near differentially expressed genes. Overall, our work demonstrates the potential of targeting protein interaction interfaces to better define the landscape of prospective phenotypes reachable by amino acid substitutions.

bioinformatics↗

bioRxiv2023

Self-delivering CRISPR RNAs for AAV Co-delivery and Genome Editing in vivo

Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a "protecting oligo"), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.

molecular biology↗