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

CRISPR/Cas12a (Cpf1) is a single RNA-guided endonuclease that provides new opportunities for targeted genome engineering through the CRISPR/Cas9 system. Only AsCpf1 have been developed for insect genome editing, and the novel Cas12a orthologs nucleases and editing efficiency require more study in insect. We compared three Cas12a orthologs nucleases, AsCpf1, FnCpf1, and LbCpf1, for their editing efficiencies and antiviral abilities in vitro. The three Cpf1 efficiently edited the BmNPV genome and inhibited BmNPV replication in BmN-SWU1 cells. The antiviral ability of the FnCpf1 system was more efficient than the SpCas9 system after infection by BmNPV. We created FnCpf1×gIE1 and SpCas9×sgIE1 transgenic hybrid lines and evaluated the gene editing efficiency of different systems at the same target site. We improved the antiviral ability using the FnCpf1 system in transgenic silkworm. This study demonstrated use of the CRISPR/Cpf1 system to achieve high editing efficiencies in the silkworm, and illustrates the use of this technology for increasing disease resistance. Author Summary Genome editing is a powerful tool that has been widely used in gene function, gene therapy, pest control, and disease-resistant engineering in most parts of pathogens research. Since the establishment of CRISPR/Cas9, powerful strategies for antiviral therapy of transgenic silkworm have emerged. Nevertheless, there is still room to expand the scope of genome editing tool for further application to improve antiviral research. Here, we demonstrate that three Cpf1 endonuclease can be used efficiency editing BmNPV genome in vitro and in vivo for the first time. More importantly, this Cpf1 system could improve the resistance of transgenic silkworms to BmNPV compare with Cas9 system, and no significant cocoons difference was observed between transgenic lines infected with BmNPV and control. These broaden the range of application of CRISPR for novel genome editing methods in silkworm and also enable sheds light on antiviral therapy.


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Introduction 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][3][4][5]. 53 Three effector proteins of class 2 type V CRISPR systems, the CRISPR/CRISPR-  research, and screening of potential targets in BmNPV infection [11,13]. We first -fold by editing the two target sites of the ie-1 gene to produce a large fragment deletion 70 [11]. The CRISPR/Cas9 gene editing technology has also been used in antiviral 71 resistance breeding by editing host factor and viral key genes in BmNPV infection. 72 However, the antiviral resistance level using this system has now reached a plateau [13, 73 16, 17].

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The CRISPR-Cas12a system (Cpf1) is a single RNA-guided endonuclease used 75 for genome editing [6]. The Cpf1 enzyme has several gene editing characteristics that 76 differ from the Cas9 system[7, 18]. One major difference between the Cpf1 and Cas9 77 systems is that Cpf1 recognizes a T-rich protospacer-adjacent motif ( 103 To determine whether the CRISPR/Cpf1 system could be used for gene editing in 104 silkworm, we examined the functionality of three Cpf1 enzymes, AsCpf1, FnCpf1 and 105 LbCpf1, which have been used to edit the genomes of mammal cells. We constructed a 106 AsCpf1, FnCpf1 and LbCpf1 expression cassette attached to nuclear localization signal 107 (NSL) and 3×HA tag, which gene is driven by the OpIE2 promoter and terminator by 108 the OpIE2-PA. The crRNA expression cassettes consisting of a 20-21-nt direct repeat 109 and a 23-nt guide sequence were arranged in tandem and driven by a signal U6 promoter 110 of B. mori. Then, we transfected BmN-SWU1 cells with individual Cpf1 orthologs and 111 gRNA to target endogenous loci containing the 5′ T-rich PAMs (Fig 1A).   inhibition effect compared with the SpCas9 system ( Fig 2B). BmNPV DNA replication 139 levels were reduced by 54.6% in the FnCpf1 system relative to the control and 140 decreased more than 38.5% compared with the SpCas9 system. We analyzed the 141 changes of VP39 protein expression in the FnCpf1 and SpCas9 systems. The Western 142 blot analysis showed that the FnCpf1 and SpCas9 system could significantly inhibit 143 VP39 protein expression. The FnCpf1 system only detected the VP39 protein at 48 h 144 p.i. (Fig 2C). After the FnCpf1 system was transfected in BmN-SWU1 cells and 145 infected with BmNPV, no significant VP39 protein expression was detected in the 146 FnCpf1 system at 0-24 h p.i.; however, a weak VP39 protein band was able to detected 147 in the SpCas9 system at 24 h p.i.. The VP39 protein expression of the FnCpf1 system 148 was also lower than that of SpCas9 system at 48 h p.i. (Fig 2C). These results 149 demonstrated that the antiviral ability of the FnCpf1 system was more effective than 150 the SpCas9 system for BmNPV at the same target site. systems were able to edit the ie-1 gene in the BmNPV genome ( Fig 3B). We also found 174 that the sequence of SpCas9 × sgIE1 lines was able to edit the target site within the 175 BmNPV genome, which mainly appeared as the absence of 330 bp, and only one 176 colony had large deletions in all sequencing ( Fig 3B). In contrast, most clones of the 177 transgenic FnCpf1 × gIE1 line showed large deletions, ranging from 500 to 1400 bp.

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To evaluate the potential off-target effects of FnCpf1, we examined all possible 180 off-target sites with high sequence similarity to gIE1 in the silkworm genomes. We 181 selected three non-specific editing sites with the highest similarity for further 182 confirmation by PCR in transgenic lines. Among the three predicted off-targeting sites, 183 we did not detect any off-target mutations in the FnCpf1 × gIE1 transgenic lines (Table   184 1). These results showed that the FnCpf1 systems used in antiviral research had no 185 significant effects on non-specific loci even for editing a highly similar site in the 186 silkworm.

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Silkworm resistance to BmNPV conferred by the CRISPR/Cpf1 system 188 We determined whether the FnCpf1 system could enhance antiviral activity  i., but the survival rate of the FnCpf1×gIE1 lines was still >65% after 10 d p. i. (Fig   197   4A). These results suggested that the CRISPR/ Cpf1 system, in transgenic silkworm, 198 could more effectively improve the antiviral activity ( Fig 4A). We determined if the 199 surviving transgenic FnCpf1 × gIE1 and SpCas9 × sgIE1 silkworm lines had altered 200 cocoon characteristics after BmNPV infection. We compared the transgenic lines to the 201 control, and found that they were similar with differences ranging from 11% to 18% 202 ( Fig 4B).  However, the viral gene expression levels increased as expected in the control (Fig 4C).  FnCpf1 was the most efficient gene editing system studied (Fig 1B). To avoid the effect 235 of the target site on gene editing efficiency and antiviral ability, we designed the target 236 site of the Cas9 system to be the same site (Fig 2A). These findings emphasize that    Table. 315 The transgenic silkworm Cas9 lines were constructed as previously reported. To presented in S1 Table. 359  corresponding PCR products were sequenced, and then aligned with the IE1 sequence.

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All of the off-target site primers used in the study are presented in S1 Table. 374 Mortality analyses 375 The OBs of BmNPV were purified from diseased larvae and stored at 4°C. The primers are listed in S1 Table. 389 Characteristics analysis of transgenic silkworm 390 The cocoon volumes of the two transgenic lines, FnCpf1×gIE1 and SpCas9×sgIE1 391 were analyzed after pupation. Each transgenic line, including 30 larvae, was 392 characterized by the mean of three independent replicates. The cocoon shell rate was 393 calculated as the combined pupa and cocoon weight.

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Statistical analysis 395 All of the data are expressed as mean ± SD of three independent experiments.

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Statistical analyses were performed with a two-sample Student's t-test using GraphPad 397 Prism 6. Differences were considered highly significant at P < 0.01.  Competing interests 412 The authors declare that they have no competing interests.  The ratios of different types of mutations.