المؤلفون: Harrington, Lucas B, Ma, Enbo, Chen, Janice S, Witte, Isaac P, Gertz, Dov, Paez-Espino, David, Al-Shayeb, Basem, Kyrpides, Nikos C, Burstein, David, Banfield, Jillian F, Doudna, Jennifer A

المصدر: Molecular Cell. 79(3)

مصطلحات موضوعية: Biochemistry and Cell Biology, Bioinformatics and Computational Biology, Biological Sciences, Infectious Diseases, Genetics, Underpinning research, 1.1 Normal biological development and functioning, Bacteria, Bacterial Proteins, Base Sequence, CRISPR-Cas Systems, Clustered Regularly Interspaced Short Palindromic Repeats, DNA, Bacterial, Endodeoxyribonucleases, Escherichia coli, Genome, Bacterial, Nucleic Acid Conformation, Phylogeny, RNA, Bacterial, RNA, Guide, Kinetoplastida, RNA, Small Untranslated, Sequence Alignment, Sequence Homology, Nucleic Acid, CRISPR-cas, Candidate Phyla Radiation (CPR) bacteria, Cas12c, Cas12d, RuvC nuclease domain, crRNA, scoutRNA, tracrRNA, Medical and Health Sciences, Developmental Biology, Biological sciences, Biomedical and clinical sciences, Health sciences

الوصف: CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/5rs657fnTest

المؤلفون: Cofsky, Joshua, Karandur, Deepti, Huang, Carolyn, Witte, Isaac, Kuriyan, John, Doudna, Jennifer

مصطلحات موضوعية: CRISPR, E. coli, R-loop, RNA, biochemistry, chemical biology, deoxyribonuclease, genome editing, Bacterial Proteins, CRISPR-Associated Proteins, CRISPR-Cas Systems, DNA, DNA Breaks, Double-Stranded, Endodeoxyribonucleases, Escherichia coli, Gene Editing, R-Loop Structures, RNA, Guide, CRISPR-Cas Systems

الوصف: Type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one strand of a double-helical DNA substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we detect intrinsic instability in DNA flanking the RNA-3 side of R-loops, which Cas12a can exploit to expose second-strand DNA for cutting. Interestingly, DNA flanking the RNA-5 side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/2d03c4w3Test

المؤلفون: Cofsky, Joshua C, Karandur, Deepti, Huang, Carolyn J, Witte, Isaac P, Kuriyan, John, Doudna, Jennifer A

مصطلحات موضوعية: Biochemistry and Cell Biology, Bioinformatics and Computational Biology, Biological Sciences, Genetics, Prevention, 1.1 Normal biological development and functioning, Underpinning research, Generic health relevance, Bacterial Proteins, CRISPR-Associated Proteins, CRISPR-Cas Systems, DNA, DNA Breaks, Double-Stranded, Endodeoxyribonucleases, Escherichia coli, Gene Editing, R-Loop Structures, RNA, Guide, Kinetoplastida, CRISPR, E. coli, R-loop, RNA, biochemistry, chemical biology, deoxyribonuclease, genome editing, Biological sciences, Biomedical and clinical sciences, Health sciences

الوصف: Type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one strand of a double-helical DNA substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we detect intrinsic instability in DNA flanking the RNA-3' side of R-loops, which Cas12a can exploit to expose second-strand DNA for cutting. Interestingly, DNA flanking the RNA-5' side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/6sq1v35sTest

المؤلفون: Davis, Jessie R., Wang, Xiao, Witte, Isaac P., Huang, Tony P., Levy, Jonathan M., Raguram, Aditya, Banskota, Samagya, Seidah, Nabil G., Musunuru, Kiran, Liu, David R.

المصدر: Nature Biomedical Engineering ; volume 6, issue 11, page 1317-1317 ; ISSN 2157-846X

مصطلحات موضوعية: Computer Science Applications, Biomedical Engineering, Medicine (miscellaneous), Bioengineering, Biotechnology

الإتاحة: https://doi.org/10.1038/s41551-022-00934-xTest
https://www.nature.com/articles/s41551-022-00934-x.pdfTest
https://www.nature.com/articles/s41551-022-00934-xTest

المؤلفون: Davis, Jessie R., Wang, Xiao, Witte, Isaac P., Huang, Tony P., Levy, Jonathan M., Raguram, Aditya, Banskota, Samagya, Seidah, Nabil G., Musunuru, Kiran, Liu, David R.

المساهمون: U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases, U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences, U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute, Howard Hughes Medical Institute, Bill and Melinda Gates Foundation

المصدر: Nature Biomedical Engineering ; volume 6, issue 11, page 1272-1283 ; ISSN 2157-846X

مصطلحات موضوعية: Computer Science Applications, Biomedical Engineering, Medicine (miscellaneous), Bioengineering, Biotechnology

الوصف: The viral delivery of base editors has been complicated by their size and by the limited packaging capacity of adeno-associated viruses (AAVs). Typically, dual-AAV approaches based on trans -splicing inteins have been used. Here we show that, compared with dual-AAV systems, AAVs with size-optimized genomes incorporating compact adenine base editors (ABEs) enable efficient editing in mice at similar or lower doses. Single-AAV-encoded ABEs retro-orbitally injected in mice led to editing efficiencies in liver (66%), heart (33%) and muscle (22%) tissues that were up to 2.5-fold those of dual-AAV ABE8e, and to a 93% knockdown (on average) of human PCSK9 and of mouse Pcsk9 and Angptl3 in circulation, concomitant with substantial reductions of plasma cholesterol and triglycerides. Moreover, three size-minimized ABE8e variants, each compatible with single-AAV delivery, collectively offer compatibility with protospacer-adjacent motifs for editing approximately 82% of the adenines in the human genome. ABEs encoded within single AAVs will facilitate research and therapeutic applications of base editing by simplifying AAV production and characterization, and by reducing the dose required for the desired level of editing.

الإتاحة: https://doi.org/10.1038/s41551-022-00911-4Test
https://www.nature.com/articles/s41551-022-00911-4.pdfTest
https://www.nature.com/articles/s41551-022-00911-4Test

المؤلفون: Harrington, Lucas B., Burstein, David, Chen, Janice S., Paez-Espino, David, Ma, Enbo, Witte, Isaac P., Cofsky, Joshua C., Kyrpides, Nikos C., Banfield, Jillian F., Doudna, Jennifer A.

المصدر: Science, 2018 Nov . 362(6416), 839-842.

الوصول الحر: https://www.jstor.org/stable/26569337Test

المؤلفون: Garst, Emma H., Lee, Hwayoung, Das, Tandrila, Bhattacharya, Shibani, Percher, Avital, Wiewiora, Rafal, Witte, Isaac P., Li, Yumeng, Peng, Tao, Im, Wonpil, Hang, Howard C.

المساهمون: Division of Molecular and Cellular Biosciences, Science, Technology and Innovation Commission of Shenzhen Municipality, National Natural Science Foundation of China, National Institute of General Medical Sciences

المصدر: ACS Chemical Biology ; volume 16, issue 5, page 844-856 ; ISSN 1554-8929 1554-8937

الإتاحة: https://doi.org/10.1021/acschembio.1c00013Test

المؤلفون: Cofsky, Joshua C, Karandur, Deepti, Huang, Carolyn J, Witte, Isaac P, Kuriyan, John, Doudna, Jennifer A

الإتاحة: https://doi.org/10.7554/elife.55143.sa2Test

المؤلفون: Harrington, Lucas B., Burstein, David, Chen, Janice S., Paez-Espino, David, Ma, Enbo, Witte, Isaac P., Cofsky, Joshua C., Kyrpides, Nikos C., Banfield, Jillian F., Doudna, Jennifer A.

مصطلحات موضوعية: 59 BASIC BIOLOGICAL SCIENCES

الوصف: CRISPR-Cas systems provide microbes with adaptive immunity to infectious nucleic acids and are widely employed as genome editing tools. These tools use RNA-guided Cas proteins whose large size (950 to 1400 amino acids) has been considered essential to their specific DNA- or RNA-targeting activities. Here we present a set of CRISPR-Cas systems from uncultivated archaea that contain Cas14, a family of exceptionally compact RNA-guided nucleases (400 to 700 amino acids). Despite their small size, Cas14 proteins are capable of targeted single-stranded DNA (ssDNA) cleavage without restrictive sequence requirements. Furthermore, target recognition by Cas14 triggers nonspecific cutting of ssDNA molecules, an activity that enables high-fidelity single-nucleotide polymorphism genotyping (Cas14-DETECTR). Metagenomic data show that multiple CRISPR-Cas14 systems evolved independently and suggest a potential evolutionary origin of single-effector CRISPR-based adaptive immunity.

وصف الملف: application/pdf

العلاقة: http://www.osti.gov/servlets/purl/1563978Test; https://www.osti.gov/biblio/1563978Test; https://doi.org/10.1126/science.aav4294Test

الإتاحة: https://doi.org/10.1126/science.aav4294Test
http://www.osti.gov/servlets/purl/1563978Test
https://www.osti.gov/biblio/1563978Test

المؤلفون: Harrington, Lucas B., Burstein, David, Chen, Janice S., Paez-Espino, David, Ma, Enbo, Witte, Isaac P., Cofsky, Joshua C., Kyrpides, Nikos C., Banfield, Jillian F., Doudna, Jennifer A.

وصف الملف: application/pdf

العلاقة: http://www.osti.gov/servlets/purl/1482289Test; https://www.osti.gov/biblio/1482289Test; https://doi.org/10.1126/science.aav4294Test

الإتاحة: https://doi.org/10.1126/science.aav4294Test
http://www.osti.gov/servlets/purl/1482289Test
https://www.osti.gov/biblio/1482289Test