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      Augmenting CRISPR applications in Drosophila with tRNA-flanked Cas9 and Cpf1 sgRNAs

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      Author(s): 1 , 3 , 1 , 3
      Publication date (Electronic):
      Journal: Nature methods

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          Abstract

          We present tRNA-based vectors for producing multiple clustered regularly-interspaced short palindromic repeats (CRISPR) sgRNAs from a single RNA polymerase II or III transcript in Drosophila. The system, which is based on liberation of sgRNAs by processing of flanking tRNAs, permits highly efficient multiplexing of Cas9-based mutagenesis. We also demonstrate that the tRNA-sgRNA system markedly increases the efficacy of conditional gene disruption by Cas9 and can promote editing by the recently discovered RNA-guided endonuclease Cpf1.

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          CRISPR RNA-guided activation of endogenous human genes

          Morgan Maeder, Samantha Linder, Vincent Cascio … (2013)
          Catalytically inactive CRISPR-associated 9 nuclease (dCas9) can be directed by short guide RNAs (gRNAs) to repress endogenous genes in bacteria and human cells. Here we show that a dCas9-VP64 transcriptional activation domain fusion protein can be directed by single or multiple gRNAs to increase expression of specific endogenous human genes. These results provide an important proof-of-principle that CRISPR-Cas systems can be used to target heterologous effector domains in human cells.
          Article 0 comments Cited 465 times – based on 0 reviews     Review now
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            Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.

            Fillip Port, Hui-Min Chen, Tzumin Lee … (2014)
            The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line-restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.
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              Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells.

              Daesik Kim, Jungeun Kim, Junho K Hur … (2016)
              Programmable clustered regularly interspaced short palindromic repeats (CRISPR) Cpf1 endonucleases are single-RNA-guided (crRNA) enzymes that recognize thymidine-rich protospacer-adjacent motif (PAM) sequences and produce cohesive double-stranded breaks (DSBs). Genome editing with CRISPR-Cpf1 endonucleases could provide an alternative to CRISPR-Cas9 endonucleases, but the determinants of targeting specificity are not well understood. Using mismatched crRNAs we found that Cpf1 could tolerate single or double mismatches in the 3' PAM-distal region, but not in the 5' PAM-proximal region. Genome-wide analysis of cleavage sites in vitro for eight Cpf1 nucleases using Digenome-seq revealed that there were 6 (LbCpf1) and 12 (AsCpf1) cleavage sites per crRNA in the human genome, fewer than are present for Cas9 nucleases (>90). Most Cpf1 off-target cleavage sites did not produce mutations in cells. We found mismatches in either the 3' PAM-distal region or in the PAM sequence of 12 off-target sites that were validated in vivo. Off-target effects were completely abrogated by using preassembled, recombinant Cpf1 ribonucleoproteins.
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                Author and article information

                Journal
                Journal ID (nlm-journal-id): 101215604
                Journal ID (pubmed-jr-id): 32338
                Journal ID (nlm-ta): Nat Methods
                Journal ID (iso-abbrev): Nat. Methods
                Title: Nature methods
                ISSN (Print): 1548-7091
                ISSN (Electronic): 1548-7105
                Publication date Nihms-submitted: 27 December 2016
                Publication date (Electronic): 05 September 2016
                Publication date (Print): October 2016
                Publication date PMC-release: 01 April 2017
                Volume: 13
                Issue: 10
                Pages: 852-854
                Affiliations
                [1 ]Cell Biology Division, MRC Laboratory of Molecular Biology, Cambridge, UK
                Author notes
                [3 ]For correspondence: Fillip Port f.port@ 123456dkfz.de , Simon Bullock sbullock@ 123456mrc-lmb.cam.ac.uk
                [2]

                Present address: Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany

                Article
                Accession ID: PMC5215823 Pmcid ID: PMC5215823 Pmc-uid ID: 5215823 Manuscript ID: ems70862
                DOI: 10.1038/nmeth.3972
                PMC ID: 5215823
                PubMed ID: 27595403
                SO-VID: 44949450-1d7a-48e2-a5f3-60d47f9ebbd4
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