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Abstract CRISPR‐Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA‐guided nuclease to initiate double‐strand breaks. Until recently, classical RAD51‐mediated homologous recombination has been a powerful tool for gene targeting in the mossPhyscomitrella patens. However, CRISPR‐Cas9‐mediated genome editing inP. patenswas shown to be more efficient than traditional homologous recombination (Plant Biotechnology Journal, 15, 2017, 122). CRISPR‐Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR‐Cas9 genome editing inP. patens, here we describe the generation and use of a flexible and modular CRISPR‐Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology‐directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5′ and 3′ ends of gene coding regions. With regard to homology‐directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a “stop codon cassette” with downstream near‐effortless genotyping.
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Whole genome sequencing data of Raoultella ornithinolytica PX02 isolated from San Jacinto river sediment in Baytown, Texas
Raoultella ornithinolytica PX02 is a Gram-negative, encapsulated bacterium, part of the Enterobacteriaceae family, emerging as a notable human pathogen. Here, we present the whole genome sequence of R. ornithinolytica PX02 isolated from San Jacinto River sediment near a Burnet Shores community in Baytown, Texas. This microorganism harbors a large 200,000 bp incF plasmid and can potentially be a significant antibiotic reservoir. The PX02 genome consists of 5,970,914 base pairs encoding approximately 5,661 functional proteins. Strain PX02 (chromosomal and plasmid) was compiled at the scaffold level and can be accessed through the National Center for Biotechnology Information database under accession NZ_NJBC00000000.1.
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- PAR ID:
- 10485224
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Data in Brief
- Volume:
- 50
- Issue:
- C
- ISSN:
- 2352-3409
- Page Range / eLocation ID:
- 109506
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/j.dib.2023.109506
