A sequential transformation method for validating soybean genome editing by CRISPR/Cas9 system

This study was performed to evaluate the sequential transformation for soybean genome editing using the CRISPR/Cas9 system as well as to show a strategy for examining the activity of CRISPR/Cas9 constructs, especially the designed guide RNAs (gRNAs). The gRNAs for targeted mutations of an exogenous gene and multiple endogenous genes were constructed and transferred into a stably-overexpressed-Cas9 soybean line using Agrobacterium rhizogenes-mediated hairy root induction system. The targeted mutations were identified and characterized by the poly-acrylamide gel electrophoresis (PAGE) heteroduplex method and by sequencing. Induced mutations of the exogenous gene (gus) were observed in 57% of tested transgenic hairy roots, while 100% of the transgenic root lines showed targeted mutations of the endogenous (SACPD-C) gene. Multiple gRNAs targeting two endogenous genes (SACPD-C and SMT) induced mutation rates of 75% and 67%, respectively. Various indels including small and large deletions as well as insertions were found in target sites of the tested genes. This sequential transformation method could present the targeting efficacy of different gRNAs of each tested gene. Additionally, in this study differences in gRNA ratings were found between bioinformatics predictions and actual experimental results. This is the first successful application of the sequential transformation method for genome editing in soybean using the hairy root system. This method could be potentially useful for validating CRISPR/Cas9 constructs, evaluating gRNA targeting efficiencies, and could be applied for other research directions.     

CRISPR/Cas9‐mediated gene knockout in the ascidian Ciona intestinalis

Knockout of genes with CRISPR/Cas9 is a newly emerged approach to investigate functions of genes in various organisms. We demonstrate that CRISPR/Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan. Short guide RNA (sgRNA) and Cas9 mRNA, when they are expressed in Ciona embryos by means of microinjection or electroporation of their expression vectors, introduced mutations in the target genes. The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on‐target site. CRISPR/Cas9‐mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases.     

Target-specific mutations efficiency at multiple loci of CRISPR/Cas9 system using one sgRNA in soybean

Soybean has a palaeopolyploid genome with nearly 75% of the genes present in multiple copies. Although the CRISPR/Cas9 system has been employed in soybean to generate site-directed mutagenesis, a systematical assessment of mutation efficiency of the CRISPR/Cas9 system for the multiple-copy genes is still urgently needed. Here, we successfully optimize one sgRNA CRISPR/Cas9 system in soybean by testing the efficiency, pattern, specificity of the mutations at multiple loci of GmFAD2 and GmALS. The results showed that simultaneous site-directed mutagenesis of two homoeologous loci by one sgRNA, the mutation frequency in the T0 generation were 64.71% for GmPDS, 60.0% for GmFAD2 and 42.86% for GmALS, respectively. The chimeric and heterozygous mutations were dominant types. Moreover, association of phenotypes with mutation pattern at target loci of GmPDS11 and GmPDS18 could help us further demonstrate that the CRISPR/Cas9 system can efficiently generate target specific mutations at multiple loci using one sgRNA in soybean, albeit with a relatively low transformation efficiency.

     

应用CRISPR/Cas9技术在杨树中高效敲除多个靶基因

CRISPR/Cas9系统是一种广泛应用于细菌、酵母、动物和植物中的基因组定点编辑技术。本课题组在前期工作中利用该系统在毛白杨(Populus tomentosa Carr.)中率先实现了对内源基因—八氢番茄红素脱氢酶(Phytoene dehydrogenase, PDS)基因的定点敲除。为研究靶点的设计和选择对该系统介导的杨树内源基因敲除效率的影响,本文分析了不同单向导RNA(Single-guide RNA, sgRNA)结合毛白杨PDS(PtPDS)靶基因DNA序列后对突变效率的影响。结果发现sgRNA与靶基因间的碱基错配会导致突变的效率降低,甚至不能突变,其中3′端的碱基配对更为重要。进一步测序分析发现,该系统能同时敲除杨树基因组上两个同源的PDS编码基因(PtPDS1和PtPDS2),突变率分别达86.4%和50%。研究证明该系统可快速高效地敲除两个以上的内源基因,获得多重突变体杨树株系。利用该技术,本课题组已获得多个杨树转录因子及结构基因的敲除突变体株系,为将来开展基因功能研究和杨树遗传改良奠定了基础。     

Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii, failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii, we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10⁹ cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii. The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing.     

Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice

The Cas9/sgRNA of the CRISPR/Cas system has emerged as a robust technology for targeted gene editing in various organisms, including plants, where Cas9/sgRNA-mediated small deletions/insertions at single cleavage sites have been reported in transient and stable transformations, although genetic transmission of edits has been reported only in Arabidopsis and rice. Large chromosomal excision between two remote nuclease-targeted loci has been reported only in a few non-plant species. Here we report in rice Cas9/sgRNA-induced large chromosomal segment deletions, the inheritance of genome edits in multiple generations and construction of a set of facile vectors for high-efficiency, multiplex gene targeting. Four sugar efflux transporter genes were modified in rice at high efficiency; the most efficient system yielding 87–100% editing in T0 transgenic plants, all with di-allelic edits. Furthermore, genetic crosses segregating Cas9/sgRNA transgenes away from edited genes yielded several genome-edited but transgene-free rice plants. We also demonstrated proof-of-efficiency of Cas9/sgRNAs in producing large chromosomal deletions (115–245 kb) involving three different clusters of genes in rice protoplasts and verification of deletions of two clusters in regenerated T0 generation plants. Together, these data demonstrate the power of our Cas9/sgRNA platform for targeted gene/genome editing in rice and other crops, enabling both basic research and agricultural applications.     

CRISPR/Cas9-mediated disruption of TaNP1 genes results in complete male sterility in bread wheat

Male sterile genes and mutants are valuable resources in hybrid seed production for monoclinous crops.High genetic redundancy due to allohexaploidy makes it difficult to obtain the nuclear recessive male sterile mutants through spontaneous mutation or chemical or physical mutagenesis methods in wheat.The emerging effective genome editing tool,CRISPR/Cas9 system,makes it possible to achieve simultaneous mutagenesis in multiple homoeoalleles.To improve the genome modification efficiency of the CRISPR/Cas9 system in wheat,we compared four different RNA polymerase(Pol) Ⅲ promoters(TaU3 p,TaU6 p,OsU3 p,and OsU6 p) and three types of sgRNA scaffold in the protoplast system.We show that the TaU3 promoter-driven optimized sgRNA scaffold was most effective.The optimized CRISPR/Cas9 system was used to edit three TaNP1 homoeoalleles,whose orthologs,OsNP1 in rice and ZmIPE1 in maize,encode a putative glucose-methanol-choline oxidoreductase and are required for male sterility.Triple homozygous mutations in TaNP1 genes result in complete male sterility.We further demonstrated that anyone wild-type copy of the three TaNP1 genes is sufficient for maintenance of male fertility.Taken together,this study provides an optimized CRISPR/Cas9 vector for wheat genome editing and a complete male sterile mutant for development of a commercially viable hybrid wheat seed production system.     

Increasing the efficiency of CRISPR‐Cas9‐VQR precise genome editing in rice

Clustered regularly interspaced short palindromic repeats‐associated protein 9 (CRISPR‐Cas9) is a revolutionary technology that enables efficient genomic modification in many organisms. Currently, the wide use of Streptococcus pyogenes Cas9 (SpCas9) primarily recognizes sites harbouring a canonical NGG protospacer adjacent motif (PAM). The newly developed VQR (D1135V/R1335Q/T1337R) variant of Cas9 has been shown to cleave sites containing NGA PAM in rice, which greatly expanded the range of genome editing. However, the low editing efficiency of the VQR variant remains, which limits its wide application in genome editing. In this study, by modifying the single guide RNA (sgRNA) structure and strong endogenous promoters, we significantly increased the editing efficiency of the VQR variant. The modified CRISPR‐Cas9‐VQR system provides a robust toolbox for multiplex genome editing at sites containing noncanonical NGA PAM.     

Cas9HF1 enhanced specificity in Ustilago maydis

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely used as a tool to precisely manipulate genomic sequence targeted by sgRNA (single guide RNA) and is adapted in different species for genome editing. One of the major concerns of CRISPR-Cas9 is the possibility of off-target effects, which can be remedied by the deployment of high fidelity Cas9 variants. Ustilago maydis is a maize fungal pathogen, which has served as a model organism for biotrophic pathogens for decades. The successful adaption of CRISPR-Cas9 in U. maydis greatly facilitated effector biology studies. Here, we constructed an U. maydis reporter strain that allows in vivo quantification of efficiency and target specificity of three high fidelity Cas9 variants, Cas9HF1, Cas9esp1.1 and Cas9hypa. This approach identified Cas9HF1 as most specific Cas9 variant in U. maydis. Furthermore, whole genome sequencing showed absence of off-target effects in U. maydis by CRISPR-Cas9 editing.     

Applications of CRISPR/Cas9 technology for targeted mutagenesis,gene replacement and stacking of genes in higher plants

Mutagenesis continues to play an essential role for understanding plant gene function and, in some instances, provides an opportunity for plant improvement. The development of gene editing technologies such as TALENs and zinc fingers has revolutionised the targeted mutation specificity that can now be achieved. The CRISPR/Cas9 system is the most recent addition to gene editing technologies and arguably the simplest requiring only two components; a small guide RNA molecule (sgRNA) and Cas9 endonuclease protein which complex to recognise and cleave a specific 20 bp target site present in a genome. Target specificity is determined by complementary base pairing between the sgRNA and target site sequence enabling highly specific, targeted mutation to be readily engineered. Upon target site cleavage, error-prone endogenous repair mechanisms produce small insertion/deletions at the target site usually resulting in loss of gene function. CRISPR/Cas9 gene editing has been rapidly adopted in plants and successfully undertaken in numerous species including major crop species. Its applications are not restricted to mutagenesis and target site cleavage can be exploited to promote sequence insertion or replacement by recombination. The multiple applications of this technology in plants are described.     

Optimized paired‐sgRNA/Cas9 cloning and expression cassette triggers high‐efficiency multiplex genome editing in kiwifruit

Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In this study, we developed a new cloning strategy for generating paired‐sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired‐sgRNA cloning, our strategy only requires the synthesis of two gRNA‐containing primers which largely reduces the cost. We further compared efficiencies of paired‐sgRNA/Cas9 vectors containing different sgRNA expression devices, including both the polycistronic tRNA‐sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system was 10‐fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired‐sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418‐resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other plants.     

Unexpected gene activation following CRISPR‐Cas9‐mediated genome editing

The discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its development as a genome editing tool has revolutionized the field of molecular biology. In the DNA damage field, CRISPR has brought an alternative to induce endogenous double‐strand breaks (DSBs) at desired genomic locations and study the DNA damage response and its consequences. Many systems for sgRNA delivery have been reported in order to efficiently generate this DSB, including lentiviral vectors. However, some of the consequences of these systems are not yet well understood. Here, we report that lentiviral‐based sgRNA vectors can integrate into the endogenous genomic target location, leading to undesired activation of the target gene. By generating a DSB in the regulatory region of the ABCB1 gene using a lentiviral sgRNA vector, we can induce the formation of Taxol‐resistant colonies. We show that these colonies upregulate ABCB1 via integration of the EEF1A1 and the U6 promoters from the sgRNA vector. We believe that this is an unreported CRISPR/Cas9 on‐target effect that researchers need to be aware of when using lentiviral vectors for genome editing.     

Patterns of CRISPR/Cas9 activity in plants,animals and microbes

The CRISPR/Cas9 system and related RNA‐guided endonucleases can introduce double‐strand breaks (DSBs) at specific sites in the genome, allowing the generation of targeted mutations in one or more genes as well as more complex genomic rearrangements. Modifications of the canonical CRISPR/Cas9 system from Streptococcus pyogenes and the introduction of related systems from other bacteria have increased the diversity of genomic sites that can be targeted, providing greater control over the resolution of DSBs, the targeting efficiency (frequency of on‐target mutations), the targeting accuracy (likelihood of off‐target mutations) and the type of mutations that are induced. Although much is now known about the principles of CRISPR/Cas9 genome editing, the likelihood of different outcomes is species‐dependent and there have been few comparative studies looking at the basis of such diversity. Here we critically analyse the activity of CRISPR/Cas9 and related systems in different plant species and compare the outcomes in animals and microbes to draw broad conclusions about the design principles required for effective genome editing in different organisms. These principles will be important for the commercial development of crops, farm animals, animal disease models and novel microbial strains using CRISPR/Cas9 and other genome‐editing tools.     

Recent advances in CRISPR/Cas9 mediated genome editing in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Genome editing using engineered nucleases has rapidly transformed from a niche technology to a mainstream method used in various host cells. Its widespread adoption has been largely developed by the emergence of the clustered regularly interspaced short palindromic repeats (CRISPR) system, which uses an easily customizable specificity RNA-guided DNA endonuclease, such as Cas9. Recently, CRISPR/Cas9 mediated genome engineering has been widely applied to model organisms, including Bacillus subtilis, enabling facile, rapid high-fidelity modification of endogenous native genes. Here, we reviewed the recent progress in B. subtilis gene editing using CRISPR/Cas9 based tools, and highlighted state-of-the-art strategies for design of CRISPR/Cas9 system. Finally, future perspectives on the use of CRISPR/Cas9 genome engineering for sequence-specific genome editing in B. subtilis are provided.     

Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in <Emphasis Type="Italic">Arabidopsis</Emphasis> and its prevention

Key message

We present novel observations of high-specificity SpCas9 variants, sgRNA expression strategies based on mutant sgRNA scaffold and tRNA processing system, and CRISPR/Cas9-mediated T-DNA integrations.

Abstract

Specificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.