scholarly journals Targeted insertion of large genetic payloads using cas directed LINE-1 reverse transcriptase

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Femila Manoj ◽  
Laura W. Tai ◽  
Katelyn Sun Mi Wang ◽  
Thomas E. Kuhlman
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcriptase Activity ◽  
Site Specific ◽  
Directed Line ◽  
Guide Rna ◽  
E Coli ◽  
Rna Targeting ◽  
Potential Strategy ◽  
Genetic Constructs ◽  
Target Effects

AbstractA difficult genome editing goal is the site-specific insertion of large genetic constructs. Here we describe the GENEWRITE system, where site-specific targetable activity of Cas endonucleases is coupled with the reverse transcriptase activity of the ORF2p protein of the human retrotransposon LINE-1. This is accomplished by providing two RNAs: a guide RNA targeting Cas endonuclease activity and an appropriately designed payload RNA encoding the desired insertion. Using E. coli as a simple platform for development and deployment, we show that with proper payload design and co-expression of helper proteins, GENEWRITE can enable insertion of large genetic payloads to precise locations, although with off-target effects, using the described approach. Based upon these results, we describe a potential strategy for implementation of GENEWRITE in more complex systems.

scholarly journals On-target activity predictions enable improved CRISPR–dCas9 screens in bacteria

2020 ◽  
Vol 48 (11) ◽  
pp. e64-e64 ◽  
Author(s):  
Alicia Calvo-Villamañán ◽  
Jérome Wong Ng ◽  
Rémi Planel ◽  
Hervé Ménager ◽  
Arthur Chen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Cost Effective ◽  
Target Sequence ◽  
Guide Rna ◽  
E Coli ◽  
Genome Wide ◽  
High Throughput Screens ◽  
Target Activity ◽  
Target Effects

Abstract The ability to block gene expression in bacteria with the catalytically inactive mutant of Cas9, known as dCas9, is quickly becoming a standard methodology to probe gene function, perform high-throughput screens, and engineer cells for desired purposes. Yet, we still lack a good understanding of the design rules that determine on-target activity for dCas9. Taking advantage of high-throughput screening data, we fit a model to predict the ability of dCas9 to block the RNA polymerase based on the target sequence, and validate its performance on independently generated datasets. We further design a novel genome wide guide RNA library for E. coli MG1655, EcoWG1, using our model to choose guides with high activity while avoiding guides which might be toxic or have off-target effects. A screen performed using the EcoWG1 library during growth in rich medium improved upon previously published screens, demonstrating that very good performances can be attained using only a small number of well designed guides. Being able to design effective, smaller libraries will help make CRISPRi screens even easier to perform and more cost-effective. Our model and materials are available to the community through crispr.pasteur.fr and Addgene.

Site-specific modification of E. coli DNA polymerase I large fragment with pyridoxal 5'-phosphate

Biochemistry ◽  
1984 ◽  
Vol 23 (9) ◽  
pp. 2073-2078 ◽  
Author(s):  
Anup K. Hazra ◽  
Sevilla Detera-Wadleigh ◽  
Samuel H. Wilson
Keyword(s):  
Dna Polymerase ◽  
Large Fragment ◽  
Dna Polymerase I ◽  
Site Specific ◽  
E Coli ◽  
Specific Modification ◽  
Polymerase I

A Method for Combined Immunoaffinity Purification and Assay of HIV-1 Reverse Transcriptase Activity Useful for Crude Samples

1996 ◽  
Vol 235 (2) ◽  
pp. 141-152 ◽  
Author(s):  
J. Lennerstrand ◽  
A.-S. Rytting ◽  
C. Örvell ◽  
J.S. Gronowitz ◽  
C.F.R. Källander
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcriptase Activity ◽  
Transcriptase Activity ◽  
Immunoaffinity Purification ◽  
Hiv 1

Application of RtcB ligase to monitor self-cleaving ribozyme activity by RNA-seq

2022 ◽  
Vol 0 (0) ◽  
Author(s):  
V. Janett Olzog ◽  
Lena I. Freist ◽  
Robin Goldmann ◽  
Jörg Fallmann ◽  
Christina E. Weinberg
Keyword(s):  
Rna Seq ◽  
Site Specific ◽  
Pyrococcus Horikoshii ◽  
Total Rna ◽  
E Coli ◽  
Catalytic Rnas ◽  
Adapter Ligation ◽  
Domains Of Life ◽  
Cyclic Phosphate ◽  
A Site

Abstract Self-cleaving ribozymes are catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5′ fragment with a 2′,3′ cyclic phosphate (2′,3′ cP) and a 3′ fragment with a 5′ hydroxyl (5′ OH) end. Recently, several strategies to enrich self-cleaving ribozymes by targeted biochemical methods have been introduced by us and others. Here, we develop an alternative strategy in which 5ʹ OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3′ phosphate of the adapter and then ligates it directly to RNAs with 5′ OH ends. Our results demonstrate that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the broadly applied Escherichia coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured a spiked 3ʹ twister ribozyme fragment from E. coli total RNA. This RNA-seq-based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5ʹ OH termini from total RNA.

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