scholarly journals Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Holly A. Rees ◽  
Alexis C. Komor ◽  
Wei-Hsi Yeh ◽  
Joana Caetano-Lopes ◽  
Matthew Warman ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Protein Delivery ◽  
Base Editing ◽  
Dna Specificity

scholarly journals Polymerase-guided base editing enables in vivo mutagenesis and rapid protein engineering

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Aaron Cravens ◽  
Osman K. Jamil ◽  
Deze Kong ◽  
Jonathan T. Sockolosky ◽  
Christina D. Smolke
Keyword(s):  
Protein Engineering ◽  
Fluorescent Protein ◽  
Random Mutagenesis ◽  
Error Rates ◽  
Lineage Tracing ◽  
T7 Promoter ◽  
Base Editing ◽  
Resistant Mutants ◽  
Essential Enzyme

AbstractRandom mutagenesis is a technique used to generate diversity and engineer biological systems. In vivo random mutagenesis generates diversity directly in a host organism, enabling applications such as lineage tracing, continuous evolution, and protein engineering. Here we describe TRIDENT (TaRgeted In vivo Diversification ENabled by T7 RNAP), a platform for targeted, continual, and inducible diversification at genes of interest at mutation rates one-million fold higher than natural genomic error rates. TRIDENT targets mutagenic enzymes to precise genetic loci by fusion to T7 RNA polymerase, resulting in mutation windows following a mutation targeting T7 promoter. Mutational diversity is tuned by DNA repair factors localized to sites of deaminase-driven mutation, enabling sustained mutation of all four DNA nucleotides at rates greater than 10−4 mutations per bp. We show TRIDENT can be applied to routine in vivo mutagenesis applications by evolving a red-shifted fluorescent protein and drug-resistant mutants of an essential enzyme.

scholarly journals Genome scale analysis of pathogenic variants targetable for single base editing

2020 ◽  
Vol 13 (S8) ◽  
Author(s):  
Alexander V. Lavrov ◽  
Georgi G. Varenikov ◽  
Mikhail Yu Skoblov
Keyword(s):  
Protein Engineering ◽  
Targeted Therapies ◽  
Human Diseases ◽  
Scale Analysis ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Single Base ◽  
Base Editing ◽  
Pathogenic Variants ◽  
Genome Scale

Abstract Background Single nucleotide variants account for approximately 90% of all known pathogenic variants responsible for human diseases. Recently discovered CRISPR/Cas9 base editors can correct individual nucleotides without cutting DNA and inducing double-stranded breaks. We aimed to find all possible pathogenic variants which can be efficiently targeted by any of the currently described base editors and to present them for further selection and development of targeted therapies. Methods ClinVar database (GRCh37_clinvar_20171203) was used to search and select mutations available for current single-base editing systems. We included only pathogenic and likely pathogenic variants for further analysis. For every potentially editable mutation we checked the presence of PAM. If a PAM was found, we analyzed the sequence to find possibility to edit only one nucleotide without changing neighboring nucleotides. The code of the script to search Clinvar database and to analyze the sequences was written in R and is available in the appendix. Results We analyzed 21 editing system currently reported in 9 publications. Every system has different working characteristics such as the editing window and PAM sequence. C > T base editors can precisely target 3196 mutations (46% of all pathogenic T > C variants), and A > G editors – 6900 mutations (34% of all pathogenic G > A variants). Conclusions Protein engineering helps to develop new enzymes with a narrower window of base editors as well as using new Cas9 enzymes with different PAM sequences. But, even now the list of mutations which can be targeted with currently available systems is huge enough to choose and develop new targeted therapies.

Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

2019 ◽  
Vol 476 (24) ◽  
pp. 3835-3847 ◽  
Author(s):  
Aliyath Susmitha ◽  
Kesavan Madhavan Nampoothiri ◽  
Harsha Bajaj
Keyword(s):  
Protein Engineering ◽  
Cell Attachment ◽  
Functional Characterization ◽  
Protein Structures ◽  
Structural Similarity ◽  
Surface Proteins ◽  
Sortase A ◽  
Peptide Cleavage ◽  
New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

Protein Engineering of Penicillin Acylase

Acta Naturae ◽  
2010 ◽  
Vol 2 (3) ◽  
pp. 47-61 ◽  
Author(s):  
V I Tishkov ◽  
S S Savin ◽  
A S Yasnaya
Keyword(s):  
Protein Engineering ◽  
Penicillin Acylase
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