CRISPR-assisted editing of bacterial genomes

By Wenyan Jiang, David Bikard, David B. T. Cox, Feng Zhang, L. Marraffini. Nature Biotechnology, 2013

1,843 citations recorded by the source corpus.

Here we use the clustered, regularly interspaced, short palindromic repeats (CRISPR)–associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relies on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems. We reprogram dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates. Simultaneous use of two crRNAs enables multiplex mutagenesis. In S. pneumoniae, nearly 100% of cells that were recovered using our approach contained the desired mutation, and in E. coli, 65% that were recovered contained the mutation, when the approach was used in combination with recombineering. We exhaustively analyze dual-RNA:Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for bacterial genome engineering.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/3f3da754cfaa1286069fdc89dbd20fe5dd130bd7

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Citations in the corpus

Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans (2014, background): “…in a wide range of organisms (Wang et al. 1999; Chiu et al. 2013; Cho et al. 2013; Dicarlo et al. 2013; Friedland et al. 2013; Gratz et al. 2013; Hwang et al. 2013; Jiang et al. 2013; Katic and Großhans 2013; Li et al. 2013; Lo et al. 2013; Nekrasov et al. 2013; Kim et al. 2014; Zhao et al. 2014).”
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Identifying and visualizing functional PAM diversity across CRISPR-Cas systems (2016, methodology): “For instance, the Streptococcus pyogenes Cas9 recognizes a weak NAG PAM in addition to the standard NGG PAM (Jiang et al., 2013b; Jinek et al., 2012), while the S. thermophilusCRISPR1Cas9 recognizesmultiple sequences that deviate from the consensus NNAGAAW PAM (where W is A or T) (Esvelt et al.,…”
Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. (2015, neutral): “By contrast, Cas9 requires both crRNA and tracrRNA to mediate targeted DNA interference (Deltcheva et al., 2011; Zhang et al., 2013).”
The CRISPR/Cas9 system for plant genome editing and beyond. (2015, background): “Subsequent work focused on additional crop species such as sorghum (Jiang et al., 2013b), wheat (Upadhyay et al., 2013; Wang et al., 2014b) andmaize (Liang et al., 2014).”
Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity (2013, background): “…can be tolerated depending on the quantity, position, and base identity of mismatches (Cong et al., 2013; Fu et al., 2013; Hsu et al., 2013; Jiang et al., 2013), leading to potential off-target (A) Cas9 with matching or mismatching sgRNA sequences targeting a locus (target 1) within the…”
Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. (2013, background): “While our paper was under review, several very recent studies reported the use of wild-type Cas9/CRISPR for targeted genome editing in diverse organisms (Mali et al., 2013; Cong et al., 2013; Jinek et al., 2013; Jiang et al., 2013; Hwang et al., 2013; Cho et al., 2013).”
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