<i>In Vitro</i>
            CRISPR/Cas9 System for Efficient Targeted DNA Editing

Liu, Yunkun; Wang, Tao; Wen, Su; Li, Zhengyuan; Yang, Anna; Deng, Zixin; Sun, Yuhui

doi:10.1128/mbio.01714-15

Cited by 61 publications

(61 citation statements)

References 44 publications

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“…The booming technology‐clustered regularly interspaced short palindromic repeats (CRISPR) system has developed rapidly over the last decade (Bayat, Modarressi, & Rahimpour, ; Ferreira, David, & Nielsen, ; Hille et al, ; Sander & Joung, ). The widely used CRISPR‐Cas9 from Streptococcus pyogenes has been designed and optimized to facilitate molecular cloning (W. Jiang et al, ; Kang, Charlop‐Powers, & Brady, ; Liu et al, ; H. Wang et al, ), such as CATCH (Cas9‐Assisted Targeting of Chromosome) (W. Jiang et al, ), ICE (in vitro CRISPR/Cas9‐mediated Editing; Liu et al, ) and exoCET (exonuclease Combined with RecET recombination; H. Wang et al, ). All these strategies are successfully used in large DNA construct cloning or modification.…”

Section: Introductionmentioning

confidence: 99%

“…The widely used CRISPR-Cas9 from Streptococcus pyogenes has been designed and optimized to facilitate molecular cloning (W. Jiang et al, 2015;Kang, Charlop-Powers, & Brady, 2016;Liu et al, 2015;H. Wang et al, 2018), such as CATCH (Cas9-Assisted Targeting of Chromosome) (W. Jiang et al, 2015), ICE (in vitro CRISPR/Cas9-mediated Editing; Liu et al, 2015) and exoCET (exonuclease Combined with RecET recombination; H. Wang et al, 2018). All these strategies are successfully used in large DNA construct cloning or modification.…”

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confidence: 99%

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Improved CRISPR‐Cas12a‐assisted one‐pot DNA editing method enables seamless DNA editing

Wang

Liu

et al. 2019

Biotech & Bioengineering

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As the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a (previously known as Cpf1) system cleaves double-stranded DNA and produces a sticky end, it could serve as a useful tool for DNA assembly/editing. To broaden its application, a variety of engineered FnCas12a proteins are generated with expanded protospacer adjacent motif (PAM) requirements. Two variants (FnCas12a-EP15 and EP16) increased the targeting range of FnCas12a by approximately fourfold. They can efficiently recognize a broad range of PAM sequences including YN (Y = C or T), TAC and CAA. Meanwhile, based on our demonstration that FnCas12a is active from 16 to 60°C, we developed an "improved CRISPR-Cas12a-assisted one-pot DNA editing" (iCOPE) method to facilitate DNA editing by combining the crRNA transcription, digestion, and ligation in one pot. By applying iCOPE, the editing efficiency reached 72-100% for two DNA fragment assemblies, and for the 21 kb large DNA construct modification, the editing efficiency can reach 100%. Thanks to the advantages of Cas12a, iCOPE with only one digestion enzyme could replace current a variety of restriction enzymes to perform the cloning in one pot with almost no sequence constraints. Taken together, this study offers an expanded DNA targeting scope of CRISPR systems and could serve as an efficient seamless one-pot DNA editing tool. K E Y W O R D SCas12a variants, DNA editing, FnCas12a, one pot, synthetic biology

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Section: Introductionmentioning

confidence: 99%

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confidence: 99%

Improved CRISPR‐Cas12a‐assisted one‐pot DNA editing method enables seamless DNA editing

Wang

Liu

et al. 2019

Biotech & Bioengineering

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“…A system called CRISPRi was recently applied to S. coelicolor , which used a catalytically dead Cas9 (dCas9) to interfere with gene expression in a sequence-specific manner [22], and thus can be potentially applied to repress transcription of negative/positive regulatory genes for discovery of natural products from silent BGCs. Other tools such as the in vitro DNA editing system [23] and the transformation-associated recombination (TAR) method [24 • ] were also established based on the versatile CRISPR/Cas9 system, which further advances technological development in natural product discovery. Successful application of the CRISPR/Cas9 system in fungi has also been reported [25] and reviewed previously [26].…”

Section: Activation Of Silent Bgcs In Native Hostsmentioning

confidence: 99%

Breaking the silence: new strategies for discovering novel natural products

Ren

Wang

Zhao

2017

Current Opinion in Biotechnology

102

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Natural products have been a prolific source of antibacterial and anticancer drugs for decades. One of the major challenges in natural product discovery is that the vast majority of natural product biosynthetic gene clusters (BGCs) have not been characterized, partially due to the fact that they are either transcriptionally silent or expressed at very low levels under standard laboratory conditions. Here we describe the strategies developed in recent years (mostly between 2014–2016) for activating silent BGCs. These strategies can be broadly divided into two categories: approaches in native hosts and approaches in heterologous hosts. In addition, we briefly discuss recent advances in developing new computational tools for identification and characterization of BGCs and high-throughput methods for detection of natural products.

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“…Compared to regular methods that deliver plasmids or mRNA encoding Cas9, increasing studies demonstrated that direct delivery of Cas9 RNP for genome editing in cells and animals has obvious advantages 17-20 , such as reduced off-target effects, low toxicity, high editing efficiency, etc. Therefore, a large amount of biopharmaceutical companies and research institutes have recently put greater emphasis on developing Cas9 RNP-based gene therapy medicines.To produce Cas9 RNP, the routine strategy needs to obtain Cas9 and sgRNAs separately and then assemble them in vitro [21][22][23] . During the process, Cas9 is generally purified by recombinant expression from E. coli, whereas sgRNAs are constructed either by in vitro transcription that often requires tedious steps, or by chemical synthesis which is time consuming and expensive.…”

mentioning

confidence: 99%

“…To produce Cas9 RNP, the routine strategy needs to obtain Cas9 and sgRNAs separately and then assemble them in vitro [21][22][23] . During the process, Cas9 is generally purified by recombinant expression from E. coli, whereas sgRNAs are constructed either by in vitro transcription that often requires tedious steps, or by chemical synthesis which is time consuming and expensive.…”

mentioning

confidence: 99%

Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly

Qiao³

et al. 2018

Preprint

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CRISPR-Cas9 is a versatile and powerful genome engineering tool. Recently, Cas9 ribonucleoprotein (RNP) complexes have been used as promising biological tools with plenty of in vivo and in vitro applications, but there are by far no efficient methods to produce Cas9 RNP at large scale and low cost. Here, we describe a simple and effective approach for direct preparation of Cas9 RNP from E. coli by co-expressing Cas9 and target specific single guided RNAs. The purified RNP showed in vivo genome editing ability, as well as in vitro endonuclease activity that combines with an superior stability to enable routine uses in molecular cloning instead of restriction enzymes. We further develop a RNP-based PCR-free method termed Cas-Brick in a one-step or cyclic for seamless assembly of multiple DNA fragments with high fidelity up to 99%. Altogether, our findings provide a general strategy to produce Cas9 RNP and supply a convenient and cost-effective DNA assembly method which is an invaluable addition to the synthetic biological toolboxes.Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated-protein 9 (Cas9) genome-editing system is a kind of the adaptive immune systems in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids 1-3 . The widely used Streptococcus pyogenes Cas9 (SpCas9) can be targeted to a 20 bp specific DNA sequence by an associated complementary guide RNA (gRNA), provided that a protospacer adjacent motif (PAM) of the form NRG (where R = G or A) is present 4,5 . The CRISPR-Cas9-based genetic technologies, in particular as genome-editing tools, have been extensively applied to biomedical area with great promises to revolutionize the treatment of genetic diseases [6][7][8][9] . Compared to other protein-based genome-editing technologies, including zinc-finger nucleases (ZFNs) 10 and transcription activator-like effector nucleases (TALENs) 11 , the newly developed RNA-guided endonucleases CRISPR-Cas9 system has attracted wide attentions due to its simplicity, high efficacy, and ease of use 8,12 . CRISPR-Cas9 has been mostly used for in vivo genome excising and editing 1,3,6 , otherwise, it and its variants such as catalytically inactivated Cas9 (dCas9) have been successfully applied in high-throughput interrogation of gene functions in health and diseases 13,14 . In spite of numerous in vivo applications, recently, CRISPR-Cas9 has been employed as in vitro molecular cloning tools as well. For instance, a technique named CATCH utilized Cas9 nuclease to excise the target genome segment up to 100 kilobases and ligated it to cloning vectors in a single step 15 . Besides, another technique called DASH harnessed Cas9 to effectively deplete unwanted sequence from DNA libraries prior to sequencing to reduce sequencing cost 16 . In these techniques, Cas9 functioned as a ribonucleprotein complex (RNP), in which the specific negative single guide RNA molecules (sgRNAs) bound to positively charged Cas9 enzymes. Compared to regular methods that deliver plasm...

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In Vitro CRISPR/Cas9 System for Efficient Targeted DNA Editing

Cited by 61 publications

References 44 publications

Improved CRISPR‐Cas12a‐assisted one‐pot DNA editing method enables seamless DNA editing

Improved CRISPR‐Cas12a‐assisted one‐pot DNA editing method enables seamless DNA editing

Breaking the silence: new strategies for discovering novel natural products

Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly

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