Ribozymes of the RNA World: Investigating Ancient Enzymes through Modern Self-Cleaving Ribozymes.

Ribozymes are RNAs that can perform catalytic functions. The discovery of ribozymes was an important milestone in science because it not only opened new avenues of research but also provided insights into the origins of life on Earth. Early life most likely used RNA as catalysts for diverse functions, however, it is unclear how RNA could have performed such diverse catalytic functions. To answer this question, my work has focused on the most abundant natural class of ribozymes in the modern world, called the self-cleaving ribozymes. They catalyze the cleavage of a specific phosphodiester bond in RNA. We investigated the different catalytic strategies used by one such ribozyme, called the hammerhead ribozyme, and uncovered how RNA can utilize tautomerization, divalent metals, and folding to diversify its chemical repertoire. To learn more about the catalytic strategies used by RNA and their biological roles, we developed a method that combines biochemistry of self-cleaving cleaving ribozymes with high-throughput sequencing to discover new self-cleaving ribozymes. New self-cleaving ribozymes will provide insights about their biological roles, and expand the catalytic toolkit available to RNA. Hence, by studying modern self-cleaving ribozymes, we can learn how ancient ribozymes catalyzed diverse chemical reactions, and the roles they may have played to sustain early life

Effectiveness of NA External Fixator with T-clamp in treating Open proximal and Distal Fractures of Tibia

Objective: To compare the outcomes of the open proximal and distal fractures of tibia treated by Nasser Awais External Fixator with T-clamp. Methodology: This cross sectional study was carried out in the department of orthopaedics Nishtar hospital, Multan in one year duration from July 2016 to July 2017. Study was started after ethical approval from hospital ethical board. Collected data of all patients was entered in SPSS software version 23 and analyzed. Mean and SD was calculated for quantitative data like age, frequency and percentages were calculated for qualitative data gender. Chi square was applied to see association of outcome variables with other variables and confounder. P value ≤ 0.05 was considered as significant. Results: Overall, there were 100% (n=180) patients; 50% (n=90) in each of the two groups. Clinical results were excellent in 88.9% (n=80) patients, good in 7.8% (n=7) patients and fair in 3.3% (n=3) patients in group A. While, in group B, the clinical results were excellent in 83.3% (n=75) patients, good in 10% (n=9) patients and fair in 6.7% (n=6) patients. No association was found between clinical results and the groups (χ2= 1.411 DF = 2, P value=0.494). Conclusion: Nasser Awais External Fixator with T-Clamp is a safe and effective technique that enhances the union rate with a low complication rate with less union time in distal tibial fracture compared to proximal fracture. Keywords: NAEF, Tibia, Open proximal, T Clamp, External Fixator.

Heavily and Fully Modified RNAs Guide Efficient SpyCas9-Mediated Genome Editing [preprint]

RNA-based drugs depend on chemical modifications to increase potency and nuclease stability, and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. No studies have yet explored chemical modification at all positions of the crRNA guide and tracrRNA cofactor. Here, we have identified several heavily-modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2\u27-OH groups) that are functional in human cells. These designs demonstrate a significant breakthrough for Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes

Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing

RNA-based drugs depend on chemical modifications to increase potency and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. Here, we explore chemical modifications at all positions of the crRNA guide and tracrRNA cofactor. We identify several heavily modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2\u27-OH groups) that are functional in human cells. These designs will contribute to Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes

Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice [preprint]

Prime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines. Using this genome modification system, we could also seed tumor formation through somatic cell editing in the adult mouse. Finally, we successfully utilize dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and demonstrate that this system enables the correction of a pathogenic mutation in the mouse liver. Our findings further establish the broad potential of this new genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction

Precision Cas9 Genome Editing in vivo with All-in-one, Self-targeting AAV Vectors [preprint]

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits, especially for large effectors like Cas9s. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. The use of compact effectors has enabled single-AAV delivery of Cas9s with 1-3 guides for edits that use end-joining repair pathways, but many precise edits that correct disease-causing mutations in vivo require homology-directed repair (HDR) templates. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs to produce segmental deletions, or a single sgRNA with an HDR template. We also examine the utility of Nme2Cas9 target sites in the vector for self-inactivation. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice. These results will enable single-vector AAVs to achieve diverse therapeutic genome editing outcomes

C-BERST: Defining subnuclear proteomic landscapes at genomic elements with dCas9-APEX2 [preprint]

Mapping proteomic composition at distinct genomic loci and subnuclear landmarks in living cells has been a long-standing challenge. Here we report that dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. By combining the spatially restricted enzymatic tagging enabled by APEX2 with programmable DNA targeting by dCas9, C-BERST has successfully identified nearly 50% of known telomere-associated factors and many known centromere-associated factors. We also identified and validated SLX4IP and RPA3 as telomeric factors, confirming C-BERST\u27s utility as a discovery platform. C-BERST enables the rapid, high-throughput identification of proteins associated with specific sequences, facilitating annotation of these factors and their roles in nuclear and chromosome biology

NmeCas9 is an intrinsically high-fidelity genome-editing platform

BACKGROUND: The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery. RESULTS: Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5\u27-N4GATT-3\u27), for NmeCas9 genome editing in human cells. CONCLUSIONS: Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering

NmeCas9 is an intrinsically high-fidelity genome editing platform [preprint]

Background: The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wild-type SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large (e.g. mammalian) genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large (~4.2 kb) open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs (e.g. from Staphylococcus aureus, Campylobacter jejuni, Geobacillus stearothermophilus and Neisseria meningitidis) are considerably smaller and therefore better suited for viral delivery. Results: Here we show that wild-type NmeCas9, when programmed with guide sequences of natural length (24 nucleotides), exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5′-N4GATT-3′), for NmeCas9 genome editing in human cells. Conclusions: Our results show that NmeCas9 is a naturally high-fidelity genome editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering