TY - JOUR
T1 - Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing
AU - Adler, Benjamin A.
AU - Hessler, Tomas
AU - Cress, Brady F.
AU - Lahiri, Arushi
AU - Mutalik, Vivek K.
AU - Barrangou, Rodolphe
AU - Banfield, Jillian
AU - Doudna, Jennifer A.
N1 - Funding Information:
We thank members of the Doudna lab, the Innovative Genomics Institute and m-CAFEs SFA for helpful discussions, encouragement and feedback. Original templates for LbuCas13a, eLbuCas13a and RfxCas13d were kindly provided by E. Charles and D. Savage. Phage SUSP1 was a generous gift from S. Adhya. We also thank H. Asahara, E. Wagner, A. Lam and R. Sachdeva for piloting custom full-plasmid sequencing and analysis pipelines that were instrumental to this study. The mature version of this pipeline is publicly accessible through the UC Berkeley Sequencing Facility. Main text figures 2 and 4 as well as Supplementary Figure 18 were created using BioRender.com. This project, B.A.A. and T.H. were supported by m-CAFEs Microbial Community Analysis & Functional Evaluation in Soils ([email protected]), a Science Focus Area led by Lawrence Berkeley National Laboratory based upon work supported by the US Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231. J.A.D. is an Investigator of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Funding Information:
We thank members of the Doudna lab, the Innovative Genomics Institute and m-CAFEs SFA for helpful discussions, encouragement and feedback. Original templates for LbuCas13a, eLbuCas13a and RfxCas13d were kindly provided by E. Charles and D. Savage. Phage SUSP1 was a generous gift from S. Adhya. We also thank H. Asahara, E. Wagner, A. Lam and R. Sachdeva for piloting custom full-plasmid sequencing and analysis pipelines that were instrumental to this study. The mature version of this pipeline is publicly accessible through the UC Berkeley Sequencing Facility. Main text figures and as well as Supplementary Figure were created using BioRender.com. This project, B.A.A. and T.H. were supported by m-CAFEs Microbial Community Analysis & Functional Evaluation in Soils ([email protected]), a Science Focus Area led by Lawrence Berkeley National Laboratory based upon work supported by the US Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231. J.A.D. is an Investigator of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/12
Y1 - 2022/12
N2 - CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against incoming RNA and DNA phages by binding to complementary target phage transcripts followed by general, non-specific RNA degradation. Here we analysed the defensive capabilities of LbuCas13a from Leptotrichia buccalis and found it to have robust antiviral activity unaffected by target phage gene essentiality, gene expression timing or target sequence location. Furthermore, we find LbuCas13a antiviral activity to be broadly effective against a wide range of phages by challenging LbuCas13a against nine E. coli phages from diverse phylogenetic groups. Leveraging the versatility and potency enabled by LbuCas13a targeting, we applied LbuCas13a towards broad-spectrum phage editing. Using a two-step phage-editing and enrichment method, we achieved seven markerless genome edits in three diverse phages with 100% efficiency, including edits as large as multi-gene deletions and as small as replacing a single codon. Cas13a can be applied as a generalizable tool for editing the most abundant and diverse biological entities on Earth.
AB - CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against incoming RNA and DNA phages by binding to complementary target phage transcripts followed by general, non-specific RNA degradation. Here we analysed the defensive capabilities of LbuCas13a from Leptotrichia buccalis and found it to have robust antiviral activity unaffected by target phage gene essentiality, gene expression timing or target sequence location. Furthermore, we find LbuCas13a antiviral activity to be broadly effective against a wide range of phages by challenging LbuCas13a against nine E. coli phages from diverse phylogenetic groups. Leveraging the versatility and potency enabled by LbuCas13a targeting, we applied LbuCas13a towards broad-spectrum phage editing. Using a two-step phage-editing and enrichment method, we achieved seven markerless genome edits in three diverse phages with 100% efficiency, including edits as large as multi-gene deletions and as small as replacing a single codon. Cas13a can be applied as a generalizable tool for editing the most abundant and diverse biological entities on Earth.
UR - http://www.scopus.com/inward/record.url?scp=85140973930&partnerID=8YFLogxK
U2 - 10.1038/s41564-022-01258-x
DO - 10.1038/s41564-022-01258-x
M3 - Article
C2 - 36316451
AN - SCOPUS:85140973930
SN - 2058-5276
VL - 7
SP - 1967
EP - 1979
JO - Nature Microbiology
JF - Nature Microbiology
IS - 12
ER -