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      Digging into the lesser-known aspects of CRISPR biology

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          Abstract

          A long time has passed since regularly interspaced DNA repeats were discovered in prokaryotes. Today, those enigmatic repetitive elements termed clustered regularly interspaced short palindromic repeats (CRISPR) are acknowledged as an emblematic part of multicomponent CRISPR-Cas (CRISPR associated) systems. These systems are involved in a variety of roles in bacteria and archaea, notably, that of conferring protection against transmissible genetic elements through an adaptive immune-like response. This review summarises the present knowledge on the diversity, molecular mechanisms and biology of CRISPR-Cas. We pay special attention to the most recent findings related to the determinants and consequences of CRISPR-Cas activity. Research on the basic features of these systems illustrates how instrumental the study of prokaryotes is for understanding biology in general, ultimately providing valuable tools for diverse fields and fuelling research beyond the mainstream.

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          Most cited references322

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          A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
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            Multiplex genome engineering using CRISPR/Cas systems.

            Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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              RNA-guided human genome engineering via Cas9.

              Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
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                Author and article information

                Contributors
                fmojica@ua.es
                Journal
                Int Microbiol
                Int Microbiol
                International Microbiology
                Springer International Publishing (Cham )
                1139-6709
                1618-1905
                6 September 2021
                6 September 2021
                2021
                : 24
                : 4
                : 473-498
                Affiliations
                [1 ]GRID grid.5268.9, ISNI 0000 0001 2168 1800, Dpto. Fisiología, Genética y Microbiología, , Universidad de Alicante, ; Alicante, Spain
                [2 ]GRID grid.5268.9, ISNI 0000 0001 2168 1800, Instituto Multidisciplinar para el Estudio del Medio, , Universidad de Alicante, ; Alicante, Spain
                Author information
                http://orcid.org/0000-0002-3789-2095
                http://orcid.org/0000-0002-6660-4996
                Article
                208
                10.1007/s10123-021-00208-7
                8616872
                34487299
                8ebe534f-3ec3-49b2-bfdb-de126da6e5fb
                © The Author(s) 2021

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 16 June 2021
                : 30 August 2021
                : 31 August 2021
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100011596, conselleria d'educació, investigació, cultura i esport;
                Award ID: PROMETEO/2017/129
                Award Recipient :
                Categories
                Review
                Custom metadata
                © Springer Nature Switzerland AG 2021

                crispr,cas proteins,adaptive immunity,rna-guided transposition,non-canonical crispr roles,crispr regulation

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