A CRISPR/LbCas12a‐based method for highly efficient multiplex gene editing in Physcomitrella patens

Due to their high efficiency, specificity, and flexibility, programmable nucleases, such as those of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (Cpf1) system, have greatly expanded the applicability of editing the genomes of various organisms. Genes from different gene families or genes with redundant functions in the same gene family can be examined by assembling multiple CRISPR RNAs (crRNAs) in a single vector. However, the activity and efficiency of CRISPR/Cas12a in the non‐vascular plant Physcomitrella patens are largely unknown. Here, we demonstrate that LbCas12a together with its mature crRNA can target multiple loci simultaneously in P. patens with high efficiency via co‐delivery of LbCas12a and a crRNA expression cassette in vivo. The mutation frequencies induced by CRISPR/LbCas12a at a single locus ranged from 26.5 to 100%, with diverse deletions being the most common type of mutation. Our method expands the repertoire of genome editing tools available for P. patens and facilitates the creation of loss‐of‐function mutants of multiple genes from different gene families.     

Genome editing in plants with MAD7 nuclease

MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1) family with a low level of homology to canonical Cas12 a nucleases. It has been publicly released as a royalty-free nuclease for both academic and commercial use. Here, we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN) in plants. Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system. We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7, as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion) system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site. Moreover, we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs. Using the CRISPR-MAD7 system, we have obtained regenerated mutant rice and wheat plants with up to 65.6% efficiency.     

Generation of targeted mutant rice using a CRISPR‐Cpf1 system

CRISPR‐Cpf1 is a newly identified CRISPR‐Cas system, and Cpf1 was recently engineered as a molecular tool for targeted genome editing in mammalian cells. To test whether the engineered CRISPR‐Cpf1 system could induce the production of rice mutants, we selected two genome targets in the OsPDS and OsBEL genes. Our results show that both targets could be efficiently mutated in transgenic rice plants using CRISPR‐Cpf1. We found that pre‐crRNAs with a full‐length direct repeat sequence exhibited considerably increased efficiencies compared with mature crRNAs. In addition, the specificity and transmission of the mutation were investigated, and the behaviours of crRNA‐Cpf1‐induced plant targeted genome mutagenesis were assessed. Taken together, our results indicate that CRISPR‐Cpf1 expression via stable transformation can efficiently generate specific and heritable targeted mutations in rice and thereby constitutes a novel and important approach to specific and precise plant genome editing.     

Targeted mutagenesis in Arabidopsis thaliana using CRISPR-Cas12b/C2c1

Cas12b/C2c1 is a newly identified class 2 CRISPR endonuclease that was recently engineered for targeted genome editing in mammals and rice. To explore the potential applications of the CRISPR‐Cas12b system in the dicot Arabidopsis thaliana, we selected BvCas12b and BhCas12b v4 for analysis. We successfully used both endonucleases to induce mutations, perform multiplex genome editing, and create large deletions at multiple loci. No significant mutations were detected at potential off‐target sites. Analysis of the insertion/deletion frequencies and patterns of mutants generated via targeted gene mutagenesis highlighted the potential utility of CRISPR‐Cas12b systems for genome editing in Arabidopsis.     

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.     

Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize

CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9‐guide RNA (gRNA) and LbCas12a‐CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium‐mediated transformation. On‐target mutation analysis showed that 90%–100% of the Cas9‐edited T0 plants carried indel mutations and 63%–77% of them were homozygous or biallelic mutants. In contrast, 0%–60% of Cas12a‐edited T0 plants had on‐target mutations. We then conducted CIRCLE‐seq analysis to identify genome‐wide potential off‐target sites for Cas9. A total of 18 and 67 potential off‐targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off‐target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.     

CRISPR家族新成员：CRISPR-Cpf1

近年来,基因组编辑技术得到了飞速发展,该技术正在基础生物学研究、医学、生物技术等多个领域引起一场新的变革.Cpf1,作为CRISPR系统的新成员,极大地扩展了基因编辑靶位点的选择范围,同时其介导的多基因编辑具有明显的优势.另外,较短的crRNA序列也使Cpf1更容易产业化.本文将从Cpf1的结构和编辑特点、应用进展、目前面临的问题及展望等方面进行介绍和总结.     

Genome Editing—Principles and Applications for Functional Genomics Research and Crop Improvement

Genome editing technologies are powerful tools for studying gene function and for crop improvement. The technologies rely on engineered endonucleases to generate double stranded breaks (DSBs) at target loci. The DSBs are repaired through the error-prone non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways in cells, resulting in mutations and sequence replacement, respectively. In the widely used CRISPR/Cas9 system, the endonuclease Cas9 is targeted by a CRISPR small RNA to DNA sequence of interest. In this review, we describe the four available types of genome editing tools, ZFN, TALEN, CRISPR/Cas9 and CRISPR/Cpf1, and show their applications in functional genomics research and precision molecular breeding of crops.     

Assembling the Streptococcus thermophilus clustered regularly interspaced short palindromic repeats (CRISPR) array for multiplex DNA targeting

In addition to the advantages of scalable, affordable, and easy to engineer, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology is superior for multiplex targeting, which is laborious and inconvenient when achieved by cloning multiple gRNA expressing cassettes. Here, we report a simple CRISPR array assembling method which will facilitate multiplex targeting usage. First, the Streptococcus thermophilus CRISPR3/Cas locus was cloned. Second, different CRISPR arrays were assembled with different crRNA spacers. Transformation assays using different Escherichia coli strains demonstrated efficient plasmid DNA targeting, and we achieved targeting efficiency up to 95% with an assembled CRISPR array with three crRNA spacers.     

谷氨酸棒杆菌中基于CRISPR/Cas9的多位点碱基编辑系统的优化

作为新型的基因组编辑工具,碱基编辑技术结合了CRISPR/Cas系统的定位功能和碱基脱氨酶的编辑功能,可实现特定位点的碱基突变,具有不产生双链DNA断裂,无需外源模板且不依赖染色体DNA同源重组的优势.目前,研究者们已在重要的工业生产菌株谷氨酸棒杆菌(Corynebacterium glutamicum)中开发了多种碱...     

Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses

Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for precise gene editing in plants. However, simultaneous gene editing of multiple homoeoalleles remains challenging, especially in self-incompatible polyploid plants. Here, we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue, using both CRISPR/Cas9 and LbCas12a (LbCpf1) systems. Loss-of-function mutants of FaPDS exhibited albino leaves, while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue. Moreover, these mutations were inheritable. Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.     

基于CRISPR/dCpf1的氧化葡萄糖酸杆菌转录抑制系统的构建与应用

氧化葡萄糖酸杆菌(Gluconobacter oxydans)因具有快速不完全氧化糖醇化合物的能力而被广泛应用于工业中.然而,适用于氧化葡萄糖酸杆菌的基因编辑工具较为缺乏,科研人员对其进行代谢改造受到很大的限制.近年来,规律成簇间隔短回文重复序列(clustered regularly interspaced shor...     

Improving CRISPR/Cas9-mediated genome editing efficiency in Yarrowia lipolytica using direct tRNA-sgRNA fusions

Yarrowia lipolytica is an important oleaginous yeast currently used in the production of specialty chemicals and has a great potential for further applications in lipid biotechnology. Harnessing the full potential of Y. lipolytica is, however, limited by its inherent recalcitrance to genetic manipulation. In contrast to Saccharomyces cerevisiae, Y. lipolytica is poor in homology-mediated DNA repair and thus in homologous recombination, which limits site-specific gene editing in this yeast. Recently developed CRISPR/Cas9-based methods using tRNA-sgRNA fusions succeeded in editing some genomic loci in Y. lipolytica. Nonetheless, the majority of other tested loci either failed editing or editing was achieved but at very low efficiency using these methods. Using tools of secondary RNA structure prediction, we were able to improve the design of the tRNA-sgRNA fusions used for the expression of single guide RNA (sgRNA) in such methods. This resulted in high efficiency CRISPR/cas9 gene editing at chromosomal loci that failed gene editing or were edited at very low efficiencies with previous methods. In addition, we characterized the gene editing performance of our newly designed tRNA-sgRNA fusions for both chromosomal gene integration and deletion. As such, this study presents an efficient CRISPR/Cas9-mediated gene-editing tool for efficient genetic engineering of Yarrowia lipolytica.     

利用CRISPR/Cas9技术对人多能干细胞进行高效基因组编辑

CRISPR/Cas9技术提供了一个全新的基因组编辑体系。本文利用CRISPR/Cas9平台,在人胚胎干细胞株中对选取的一段特定基因组区域进行了多种基因组编辑：通过在基因编码框中引入移码突变进行基因敲除;通过单链DNA提供外源模板经由同源重组定点敲入FLAG序列;通过同时靶向多个位点诱导基因组大片段删除。研究结果表明CRISPR/Cas9可以对多能干细胞进行高效基因编辑,获得的突变干细胞株有助于对基因和基因组区域的功能进行分析和干细胞疾病模型的建立。     

CRISPR‐LbCas12a‐mediated modification of citrus

Recently, CRISPR‐Cas12a (Cpf1) from Prevotella and Francisella was engineered to modify plant genomes. In this report, we employed CRISPR‐LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time. First, LbCas12a was used to modify the CsPDS gene successfully in Duncan grapefruit via Xcc‐facilitated agroinfiltration. Next, LbCas12a driven by either the 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter (EBE_PthA4‐CsLOBP) of CsLOB1. A single crRNA was selected to target a conserved region of both Type I and Type II CsLOBPs, since the protospacer adjacent motif of LbCas12a (TTTV) allows crRNA to act on the conserved region of these two types of CsLOBP. CsLOB1 is the canker susceptibility gene, and it is induced by the corresponding pathogenicity factor PthA4 in Xanthomonas citri by binding to EBE_PthA4‐CsLOBP. A total of seven 35S‐LbCas12a‐transformed Duncan plants were generated, and they were designated as #D₃₅s1 to #D₃₅s7, and ten Yao‐LbCas12a‐transformed Duncan plants were created and designated as #D_yao1 to #D_yao10. LbCas12a‐directed EBE_PthA4‐CsLOBP modifications were observed in three 35S‐LbCas12a‐transformed Duncan plants (#D₃₅s1, #D₃₅s4 and #D₃₅s7). However, no LbCas12a‐mediated indels were observed in the Yao‐LbCas12a‐transformed plants. Notably, transgenic line #D₃₅s4, which contains the highest mutation rate, alleviates XccΔpthA4:dCsLOB1.4 infection. Finally, no potential off‐targets were observed. Therefore, CRISPR‐LbCas12a can readily be used as a powerful tool for citrus genome editing.     

Data Comparison and Software Design for Easy Selection and Application of CRISPR-based Genome Editing Systems in Plants

CRISPR-based genome editing systems have been successfully and effectively used in many organisms. However, only a few studies have reported the comparison between CRISPR/Cas9 and CRISPR/Cpf1 systems in the whole-genome applications. Although many web-based toolkits are available, there is still a shortage of comprehensive, user-friendly, and plant-specific CRISPR databases and desktop software. In this study, we identified and analyzed the similarities and differences between CRISPR/Cas9 and CRISPR/Cpf1 systems by considering the abundance of proto-spacer adjacent motif (PAM) sites, the effects of GC content, optimal proto-spacer length, potential universality within the plant kingdom, PAM-rich region (PARR) inhibiting ratio, and the effects of G-quadruplex (G-Q) structures. Using this information, we built a comprehensive CRISPR database (including 138 plant genome data sources, www.grapeworld.cn/pc/index.html), which provides search tools for the identification of CRISPR editing sites in both CRISPR/Cas9 and CRISPR/Cpf1 systems. We also developed a desktop software on the basis of the Perl/Tk tool, which facilitates and improves the detection and analysis of CRISPR editing sites at the whole-genome level on Linux and/or Windows platform. Therefore, this study provides helpful data and software for easy selection and application of CRISPR-based genome editing systems in plants.