Single plasmid construction for development CRISPR-Cpf1 in A. aculeatus TBRC 277 v1 (protocols.io.bhv6j69e)

protocols.io ◽  
2020 ◽  
Author(s):  
Dede Abdulrachman ◽  
Kusol Pootanakit ◽  
Duriya Chantasingh
1998 ◽  
Vol 259 (2) ◽  
pp. 187-194 ◽  
Author(s):  
Qi Zeng ◽  
Y.H. Tan ◽  
Wanjin Hong

2002 ◽  
Vol 46 (8) ◽  
pp. 2337-2343 ◽  
Author(s):  
Julien Haroche ◽  
Jeanine Allignet ◽  
Névine El Solh

ABSTRACT We characterized a new transposon, Tn5406 (5,467 bp), in a clinical isolate of Staphylococcus aureus (BM3327). It carries a variant of vgaA, which encodes a putative ABC protein conferring resistance to streptogramin A but not to mixtures of streptogramins A and B. It also carries three putative genes, the products of which exhibit significant similarities (61 to 73% amino acid identity) to the three transposases of the staphylococcal transposon Tn554. Like Tn554, Tn5406 failed to generate target repeats. In BM3327, the single copy of Tn5406 was inserted into the chromosomal att554 site, which is the preferential insertion site of Tn554. In three other independent S. aureus clinical isolates, Tn5406 was either present as a single plasmid copy (BM3318), as two chromosomal copies (BM3252), or both in the chromosome and on a plasmid (BM3385). The Tn5406-carrying plasmids also contain two other genes, vgaB and vatB. The insertion sites of Tn5406 in BM3252 were studied: one copy was in att554, and one copy was in the additional SCCmec element. Amplification experiments revealed circular forms of Tn5406, indicating that this transposon might be active. To our knowledge, a transposon conferring resistance to streptogramin A and related compounds has not been previously described.


2017 ◽  
Vol 5 (35) ◽  
Author(s):  
Rachel J. M. Brewer ◽  
Timothy L. Haskett ◽  
Joshua P. Ramsay ◽  
Graham W. O’Hara ◽  
Jason J. Terpolilli

ABSTRACT We report here the complete genome sequence of Mesorhizobium ciceri bv. biserrulae strain WSM1497, the efficient nitrogen-fixing microsymbiont and commercial inoculant in Australia of the forage legume Biserrula pelecinus. The genome consists of 7.2 Mb distributed across a single chromosome (6.67 Mb) and a single plasmid (0.53 Mb).


Archaea ◽  
2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Shoji Suzuki ◽  
Norio Kurosawa

Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA) by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER). In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr-) and arginine decarboxylase- (argD-) deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.


2021 ◽  
Author(s):  
Lijie Wang ◽  
Wei Xue ◽  
Hongxia Zhang ◽  
Runze Gao ◽  
Houyuan Qiu ◽  
...  

Abstract Fusion of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications but suffers from off-target (OT) mutations. Here, we applied a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. This step-by-step protocol describes the plasmid construction of tBE system, determination of genome/transcriptome-wide OT mutations and tBE-mediated base editing in vivo.


2016 ◽  
Vol 4 (3) ◽  
Author(s):  
Timothy Haskett ◽  
Penghao Wang ◽  
Joshua Ramsay ◽  
Graham O’Hara ◽  
Wayne Reeve ◽  
...  

We report the complete genome sequence of Mesorhizobium ciceri bv. biserrulae strain WSM1284, a nitrogen-fixing microsymbiont of the pasture legume Biserrula pelecinus . The genome consists of 6.88 Mb distributed between a single chromosome (6.33 Mb) and a single plasmid (0.55 Mb).


2018 ◽  
Vol 163 (5) ◽  
pp. 391-398 ◽  
Author(s):  
Yi Yang ◽  
Min You ◽  
Fentian Chen ◽  
Tianrong Jia ◽  
Yuanzhi Chen ◽  
...  

2020 ◽  
Vol 9 (3) ◽  
Author(s):  
Hallie E. Rauch ◽  
Julie Haendiges ◽  
Maria Balkey ◽  
Maria Hoffmann

We report here the closed genomes of Salmonella enterica strains from the 2017–2018 multistrain, multistate kratom outbreak using single-molecule real-time DNA sequencing. Four of the genomes consist of one circular chromosome, and the fifth has a circular chromosome and a single plasmid.


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