CRISPR-Cas3 induces broad and unidirectional genome editing in human cells

Morisaka, Hiroyuki; Yoshimi, Kazuto; Okuzaki, Yuya; Gee, Peter; Kunihiro, Yayoi; Sonpho, Ekasit; Xu, Huaigeng; Sasakawa, Nobuyuki; Naito, Yukiko; Nakada, Shinichiro; Yamamoto, Takashi; Sano, Shigetoshi; Hotta, Akitsu; Takeda, Junji; Mashimo, Tomoji

doi:10.1038/s41467-019-13226-x

Cited by 135 publications

(125 citation statements)

References 73 publications

(118 reference statements)

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“…Remarkably, we detected primed adaptation over a distance of >100 kb upstream of each of the two protospacers, with the extent of primed adaptation decreasing as a function of distance from the protospacer, albeit with considerable local variation ( Figure 3B). Of note, recent studies described priming on the E. coli chromosome over a similar distance (Shiriaeva et al, 2019), and unidirectional deletions over distances up to 100 kb upon expression of type I-E CRISPR-Cas systems in human cells (Dolan et al, 2019;Morisaka et al, 2019). Our data strongly suggest that Cas3 can translocate >100 kb from the protospacer during primed adaptation.…”

Section: Resultssupporting

confidence: 64%

Characterization of Primed Adaptation in the Escherichia coli type I-E CRISPR-Cas System

Stringer

Cooper

Kadaba

et al. 2020

Preprint

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CRISPR-Cas systems are bacterial immune systems that target invading nucleic acid. The hallmark of CRISPR-Cas systems is the CRISPR array, a genetic locus that includes short sequences known as “spacers”, that are derived from invading nucleic acid. Upon exposure to an invading nucleic acid molecule, bacteria/archaea with functional CRISPR-Cas systems can add new spacers to their CRISPR arrays in a process known as “adaptation”. In type I CRISPR-Cas systems, which represent the majority of CRISPR-Cas systems found in nature, adaptation can occur by two mechanisms: naïve and primed. Here, we show that, for the archetypal type I-E CRISPR-Cas system from Escherichia coli, primed adaptation occurs at least 1,000 times more efficiently than naïve adaptation. By initiating primed adaptation on the E. coli chromosome, we show that spacers can be acquired across distances of >100 kb from the initially targeted site, and we identify multiple factors that influence the efficiency with which sequences are acquired as new spacers. Thus, our data provide insight into the mechanism of primed adaptation.

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Section: Resultssupporting

confidence: 64%

Characterization of Primed Adaptation in the Escherichia coli type I-E CRISPR-Cas System

Stringer

Cooper

Kadaba

et al. 2020

Preprint

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“…Genome editing has the potential to accurately edit the genomes of model organism [2, 20, 22]. Cas9, Cas12a (Cpf1), Cas12b, Cas13, Cas3 and Cas14 based CRISPR systems have been explored for editing human, animals, plants and microbe genomes[23-28]. Cpf1 is a type V CRISPR-effector protein with greater specificity for genome editing in mammals and plants[6, 7].…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cpf1 mediated genome editing enhances Bombyx mori resistance to BmNPV

Dong

et al. 2020

Preprint

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18CRISPR/Cas12a (Cpf1) is a single RNA-guided endonuclease that provides new 19 opportunities for targeted genome engineering through the CRISPR/Cas9 system. Only 20 AsCpf1 have been developed for insect genome editing, and the novel Cas12a orthologs 21 nucleases and editing efficiency require more study in insect. We compared three 22 Cas12a orthologs nucleases, AsCpf1, FnCpf1, and LbCpf1, for their editing efficiencies 23 and antiviral abilities in vitro. The three Cpf1 efficiently edited the BmNPV genome 24 and inhibited BmNPV replication in BmN-SWU1 cells. The antiviral ability of the 25 FnCpf1 system was more efficient than the SpCas9 system after infection by BmNPV. 26 We created FnCpf1×gIE1 and SpCas9×sgIE1 transgenic hybrid lines and evaluated the 27 gene editing efficiency of different systems at the same target site. We improved the 28 antiviral ability using the FnCpf1 system in transgenic silkworm. This study 29 demonstrated use of the CRISPR/Cpf1 system to achieve high editing efficiencies in 30 the silkworm, and illustrates the use of this technology for increasing disease resistance. 31Genome editing is a powerful tool that has been widely used in gene function, gene 36 therapy, pest control, and disease-resistant engineering in most parts of pathogens 37 research. Since the establishment of CRISPR/Cas9, powerful strategies for antiviral 38 therapy of transgenic silkworm have emerged. Nevertheless, there is still room to 39 expand the scope of genome editing tool for further application to improve antiviral 40 research. Here, we demonstrate that three Cpf1 endonuclease can be used efficiency 41 editing BmNPV genome in vitro and in vivo for the first time. More importantly, this 42 Cpf1 system could improve the resistance of transgenic silkworms to BmNPV compare 43 with Cas9 system, and no significant cocoons difference was observed between 44 transgenic lines infected with BmNPV and control. These broaden the range of 45 application of CRISPR for novel genome editing methods in silkworm and also enable 46 sheds light on antiviral therapy.47 49 Genome editing introduces DNA mutations in the form of insertions, deletions or 50 base substitutions within selected DNA sequences [1]. Clustered regularly interspaced 51 short palindromic repeats (CRISPR) gene editing technology has been used in gene 52 function research, genetic improvement, modelling biology and gene therapy [2-5]. 53 Three effector proteins of class 2 type V CRISPR systems, the CRISPR/CRISPR-54 associated 12a (Cas12a, known as Cpf1) proteins of Lachnosperaceae bacterium 55 (LbCpf1), Francisella novicida (FnCpf1) and Acidaminoccocus sp. (AsCpf1), have 56 been shown to efficiently edit mammalian cell genomes with more efficient genome 57 editing than the widely used Streptococcus pyogenes Cas9 (SpCas9) [6-8]. However, 58 the CRISPR/Cpf1 system was rarely used for insect genome editing research and 59 antiviral therapy [9].60 The silk industry (Bombyx mori, B. mori), suffers great economic losses due to B. 61 mori nucleopolyhedr...

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“…Previous studies have shown that the 6th nucleotide of the protospacer is not engaged in base pairing with the target DNA strand, because every 6th nucleotide binds with the thumb domain of Cas7 subunits in the Cascade complex (Jackson et al 2014;Mulepati et al 2014;Zhao et al 2014;Xiao et al 2018). The obvious difference between TiD and type I-E was the effect of a mismatch at 1-nt, where the DNA cleavage activity in the type I-E system is markedly reduced (Morisaka et al 2019), suggesting that the mode and/or position of RNA-Cas7 binding might differ between the two systems. In addition, no homolog of small subunit Cse2 in type I-E was found in the TiD locus.…”

Section: Impact On Tid Activity Of Mismatch Sequence Between Protospamentioning

confidence: 99%

“…The CRISPR systems, especially, Class 2 type II systems [CRISPR/Cas9 (Cong et al, 2013;Mali et al, 2013)] and type V [CRISPR/CpfI (Zetsche et al 2015)] have been widely used to efficiently introduce a mutation on a target DNA of interest in eukaryotic chromosomal DNA as a tool of genome editing. Although less common than Class 2 systems, Class 1 type I CRISPR systems have some advantages compared to Cas9 and Cpf1 as genome editing tools, such as a large variety of mutation profiles, including longer gRNA sequences, which might allow higher specificity and longrange genome deletion (Cameron et al 2019;Dolan et al 2019;Morisaka et al 2019).…”

Section: Introductionmentioning

confidence: 99%

Genome editing in mammals using CRISPR type I-D nuclease

Osakabe

Wada

Murakami

et al. 2020

Preprint

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Adoption of the CRISPR-Cas system has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I-the most abundant CRISPR system in bacteriahas been less developed. Type I systems in which Cas3 nuclease degrades the target DNA are known; in contrast, for the sub-type CRISPR type I-D (TiD), which lacks a typical Cas3 nuclease in its cascade, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d-a nuclease in TiD-is multi-functional in PAM recognition, stabilization and target DNA degradation. TiD can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. TiD off-target effects, which were dependent on the mismatch position in the protospacer of TiD, were also identified. Our findings suggest TiD as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.

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CRISPR-Cas3 induces broad and unidirectional genome editing in human cells

Cited by 135 publications

References 73 publications

Characterization of Primed Adaptation in the Escherichia coli type I-E CRISPR-Cas System

Characterization of Primed Adaptation in the Escherichia coli type I-E CRISPR-Cas System

CRISPR/Cpf1 mediated genome editing enhances Bombyx mori resistance to BmNPV

Genome editing in mammals using CRISPR type I-D nuclease

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