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CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering
Existing technologies for site-specific integration of kilobase-sized DNA sequences in bacteria are limited by low efficiency, a reliance on recombination, the need for multiple vectors, and challenges in multiplexing. To address these shortcomings, we introduce a substantially improved version of o...
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| Published in: | Nature biotechnology 2021-04, Vol.39 (4), p.480-489 |
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| container_title | Nature biotechnology |
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| creator | Vo, Phuc Leo H. Ronda, Carlotta Klompe, Sanne E. Chen, Ethan E. Acree, Christopher Wang, Harris H. Sternberg, Samuel H. |
| description | Existing technologies for site-specific integration of kilobase-sized DNA sequences in bacteria are limited by low efficiency, a reliance on recombination, the need for multiple vectors, and challenges in multiplexing. To address these shortcomings, we introduce a substantially improved version of our previously reported Tn
7
-like transposon from
Vibrio cholerae
, which uses a Type I-F CRISPR–Cas system for programmable, RNA-guided transposition. The optimized insertion of transposable elements by guide RNA–assisted targeting (INTEGRATE) system achieves highly accurate and marker-free DNA integration of up to 10 kilobases at ~100% efficiency in bacteria. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplexed insertions in three genomic loci and facile, multi-loci deletions by combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in biomedically and industrially relevant bacteria and achieved target- and species-specific integration in a complex bacterial community. This work establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering.
Optimized RNA-guided transposons efficiently integrate large DNA sequences in multiple bacterial species. |
| doi_str_mv | 10.1038/s41587-020-00745-y |
| format | article |
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7
-like transposon from
Vibrio cholerae
, which uses a Type I-F CRISPR–Cas system for programmable, RNA-guided transposition. The optimized insertion of transposable elements by guide RNA–assisted targeting (INTEGRATE) system achieves highly accurate and marker-free DNA integration of up to 10 kilobases at ~100% efficiency in bacteria. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplexed insertions in three genomic loci and facile, multi-loci deletions by combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in biomedically and industrially relevant bacteria and achieved target- and species-specific integration in a complex bacterial community. This work establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering.
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7
-like transposon from
Vibrio cholerae
, which uses a Type I-F CRISPR–Cas system for programmable, RNA-guided transposition. The optimized insertion of transposable elements by guide RNA–assisted targeting (INTEGRATE) system achieves highly accurate and marker-free DNA integration of up to 10 kilobases at ~100% efficiency in bacteria. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplexed insertions in three genomic loci and facile, multi-loci deletions by combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in biomedically and industrially relevant bacteria and achieved target- and species-specific integration in a complex bacterial community. This work establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering.
Optimized RNA-guided transposons efficiently integrate large DNA sequences in multiple bacterial species.</description><subject>631/337</subject><subject>631/61</subject><subject>Agriculture</subject><subject>Bacteria</subject><subject>Bioinformatics</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedical Engineering/Biotechnology</subject><subject>Biomedicine</subject><subject>Biotechnology</subject><subject>Control</subject><subject>CRISPR</subject><subject>CRISPR-Cas Systems</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA Transposable Elements</subject><subject>Efficiency</subject><subject>Gene Editing - methods</subject><subject>Gene sequencing</subject><subject>Gene therapy</subject><subject>Genome editing</subject><subject>Genome, Bacterial</subject><subject>Genomes</subject><subject>Integrases</subject><subject>Life Sciences</subject><subject>Loci</subject><subject>Mathematical analysis</subject><subject>Methods</subject><subject>Microbial genetic engineering</subject><subject>Multiplexing</subject><subject>Nucleotide sequence</subject><subject>Plasmids - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nature biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Vo, Phuc Leo H.</au><au>Ronda, Carlotta</au><au>Klompe, Sanne E.</au><au>Chen, Ethan E.</au><au>Acree, Christopher</au><au>Wang, Harris H.</au><au>Sternberg, Samuel H.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering</atitle><jtitle>Nature biotechnology</jtitle><stitle>Nat Biotechnol</stitle><addtitle>Nat Biotechnol</addtitle><date>2021-04-01</date><risdate>2021</risdate><volume>39</volume><issue>4</issue><spage>480</spage><epage>489</epage><pages>480-489</pages><issn>1087-0156</issn><eissn>1546-1696</eissn><abstract>Existing technologies for site-specific integration of kilobase-sized DNA sequences in bacteria are limited by low efficiency, a reliance on recombination, the need for multiple vectors, and challenges in multiplexing. To address these shortcomings, we introduce a substantially improved version of our previously reported Tn
7
-like transposon from
Vibrio cholerae
, which uses a Type I-F CRISPR–Cas system for programmable, RNA-guided transposition. The optimized insertion of transposable elements by guide RNA–assisted targeting (INTEGRATE) system achieves highly accurate and marker-free DNA integration of up to 10 kilobases at ~100% efficiency in bacteria. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplexed insertions in three genomic loci and facile, multi-loci deletions by combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in biomedically and industrially relevant bacteria and achieved target- and species-specific integration in a complex bacterial community. This work establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering.
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| ispartof | Nature biotechnology, 2021-04, Vol.39 (4), p.480-489 |
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| source | Nature |
| subjects | 631/337 631/61 Agriculture Bacteria Bioinformatics Biomedical and Life Sciences Biomedical Engineering/Biotechnology Biomedicine Biotechnology Control CRISPR CRISPR-Cas Systems Deoxyribonucleic acid DNA DNA Transposable Elements Efficiency Gene Editing - methods Gene sequencing Gene therapy Genome editing Genome, Bacterial Genomes Integrases Life Sciences Loci Mathematical analysis Methods Microbial genetic engineering Multiplexing Nucleotide sequence Plasmids - genetics Recombination Ribonucleic acid RNA RNA, Guide, CRISPR-Cas Systems - genetics Transposition Transposons Vibrio cholerae - genetics |
| title | CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering |
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