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.     

High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system

Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologues. To efficiently achieve site/gene‐specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has exerted broad site‐specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2) and an endogenous gene GhCLA1 were chosen as targets. The DsRed2‐edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7–100%. The mutation genotype was checked for both genes with Sanger sequencing. Barcode‐based high‐throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1‐edited T0 plants and it matched well with Sanger sequencing results. No off‐target editing was detected at the potential off‐target sites. These results prove that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing.     

High‐efficient and precise base editing of C•G to T•A in the allotetraploid cotton (Gossypium hirsutum) genome using a modified CRISPR/Cas9 system

The base‐editing technique using CRISPR/nCas9 (Cas9 nickase) or dCas9 (deactivated Cas9) fused with cytidine deaminase is a powerful tool to create point mutations. In this study, a novel G. hirsutum‐Base Editor 3 (GhBE3) base‐editing system has been developed to create single‐base mutations in the allotetraploid genome of cotton (Gossypium hirsutum). A cytidine deaminase sequence (APOBEC) fused with nCas9 and uracil glycosylase inhibitor (UGI) was inserted into our CRISPR/Cas9 plasmid (pRGEB32‐GhU6.7). Three target sites were chosen for two target genes, GhCLA and GhPEBP, to test the efficiency and accuracy of GhBE3. The editing efficiency ranged from 26.67 to 57.78% at the three target sites. Targeted deep sequencing revealed that the C→T substitution efficiency within an ‘editing window’, approximately six‐nucleotide windows of ?17 to ?12 bp from the PAM sequence, was up to 18.63% of the total sequences. The 27 most likely off‐target sites predicted by CRISPR‐P and Cas‐OFFinder tools were analysed by targeted deep sequencing, and it was found that rare C→T substitutions (average < 0.1%) were detected in the editing windows of these sites. Furthermore, whole‐genome sequencing analyses on two GhCLA‐edited and one wild‐type plants with about 100× depth showed that no bona fide off‐target mutations were detectable from 1500 predicted potential off‐target sites across the genome. In addition, the edited bases were inherited to T1 progeny. These results demonstrate that GhBE3 has high specificity and accuracy for the generation of targeted point mutations in allotetraploid cotton.     

Whole genome sequencing reveals rare off‐target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9‐edited cotton plants

The CRISPR/Cas9 system has been extensively applied for crop improvement. However, our understanding of Cas9 specificity is very limited in Cas9‐edited plants. To identify on‐ and off‐target mutation in an edited crop, we described whole genome sequencing (WGS) of 14 Cas9‐edited cotton plants targeted to three genes, and three negative (Ne) control and three wild‐type (WT) plants. In total, 4188–6404 unique single‐nucleotide polymorphisms (SNPs) and 312–745 insertions/deletions (indels) were detected in 14 Cas9‐edited plants compared to WT, negative and cotton reference genome sequences. Since the majority of these variations lack a protospacer‐adjacent motif (PAM), we demonstrated that the most variations following Cas9‐edited are due either to somaclonal variation or/and pre‐existing/inherent variation from maternal plants, but not off‐target effects. Of a total of 4413 potential off‐target sites (allowing ≤5 mismatches within the 20‐bp sgRNA and 3‐bp PAM sequences), the WGS data revealed that only four are bona fide off‐target indel mutations, validated by Sanger sequencing. Moreover, inherent genetic variation of WT can generate novel off‐target sites and destroy PAMs, which suggested great care should be taken to design sgRNA for the minimizing of off‐target effect. These findings suggested that CRISPR/Cas9 system is highly specific for cotton plants.     

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.     

Cas9蛋白变体VQR高效识别水稻NGAC前间区序列邻近基序

成簇的规律间隔短回文重复序列及CRISPR相关蛋白(clustered regularly interspaced short palindromic repeats/CRISPR-associated 9, CRISPR/Cas9)系统是近年来发展起来并被广泛应用的第三代基因组编辑工具。但是,该系统的酿脓链球菌Cas9(Streptococcus pyogenes, SpCas9)仅能识别NGG前间区序列邻近基序(protospacer adjacent motif, PAM),极大地限制了基因组编辑的范围。SpCas9变体VQR(D1135V/R1335Q/T1337R)在水稻中可识别NGAA、NGAG和NGAT PAM,但尚不清楚是否能识别NGAC PAM。本研究利用改进后的CRISPR/VQR系统对水稻中3个相对低效的VQR靶位点NAL1-Q1、NAL1-Q2和LPA1-Q进行了编辑,结果表明改进后的CRISPR/VQR系统可以高效编辑这3个靶位点,编辑效率分别为9.75%、43.90%和29.26%。为了明确改进后的CRISPR/VQR系统对NGAC PAM的识别情况,本研究选择水稻叶片宽度调控基因NARROW LEAF 1 (NAL1)中的NAL-C位点和蜡质合成基因GLOSSY1 (GL1)中的GL1-C位点进行基因编辑,并获得57株转基因水稻。靶位点PCR扩增及测序结果表明,NAL1-C和GL1-C靶标位点突变的植株分别为27株和44株,突变率分别为47.36%和77.19%;其中NAL1-C/GL1-C双突变植株为26株,双突变率为45.61%。进一步分析表明,CRISPR/VQR系统造成的突变有4种类型,分别为杂合突变、双等位突变、嵌合体突变和纯合突变,其中以杂合突变和双等位突变为主。这些结果表明,改进的CRISPR/VQR系统可以高效编辑水稻NGAC PAM位点,并产生丰富的突变类型。本研究为水稻及其他植物相关基因NGAC PAM位点的编辑提供了理论依据。     

Multiplexed CRISPR/Cas9‐mediated metabolic engineering of γ‐aminobutyric acid levels in Solanum lycopersicum

In recent years, the type II CRISPR system has become a widely used and robust technique to implement site‐directed mutagenesis in a variety of species including model and crop plants. However, few studies manipulated metabolic pathways in plants using the CRISPR system. Here, we introduced the pYLCRISPR/Cas9 system with one or two single‐site guide RNAs to target the tomato phytoene desaturase gene. An obvious albino phenotype was observed in T0 regenerated plants, and more than 61% of the desired target sites were edited. Furthermore, we manipulated the γ‐aminobutyric acid (GABA) shunt in tomatoes using a multiplex pYLCRISPR/Cas9 system that targeted five key genes. Fifty‐three genome‐edited plants were obtained following single plant transformation, and these samples represented single to quadruple mutants. The GABA accumulation in both the leaves and fruits of genomically edited lines was significantly enhanced, and the GABA content in the leaves of quadruple mutants was 19‐fold higher than that in wild‐type plants. Our data demonstrate that the multiplex CRISPR/Cas9 system can be exploited to precisely edit tomato genomic sequences and effectively create multisite knockout mutations, which could shed new light on plant metabolic engineering regulations.     

Development of an Agrobacterium‐delivered CRISPR/Cas9 system for wheat genome editing

CRISPR/Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR/Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR/Cas9 complex. Due to the high frequency of gene silencing associated with co‐transferred plasmid backbone and low edit rate in wheat, a large T₀ transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium‐delivered CRISPR/Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR/Cas9 system, we have developed 68 edit mutants for four grain‐regulatory genes, TaCKX2‐1, TaGLW7, TaGW2, and TaGW8, in T₀, T₁, and T₂ generation plants at an average edit rate of 10% without detecting off‐target mutations in the most Cas9‐active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160‐bp deletion in TaCKX2‐D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium‐delivered CRISPR/Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR/Cas9 remains active for novel mutations through generations.     

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.     

Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated endonuclease 9(CRISPR/Cas9) system has emerged as a promising technology for specific genome editing in many species. Here we constructed one vector targeting eight agronomic genes in rice using the CRISPR/Cas9 multiplex genome editing system. By subsequent genetic transformation and DNA sequencing, we found that the eight target genes have high mutation efficiencies in the T_0 generation. Both heterozygous and homozygous mutations of all editing genes were obtained in T_0 plants. In addition, homozygous sextuple, septuple, and octuple mutants were identified. As the abundant genotypes in T_0 transgenic plants, various phenotypes related to the editing genes were observed. The findings demonstrate the potential of the CRISPR/Cas9 system for rapid introduction of genetic diversity during crop breeding.     

Establishment of an efficient seed fluorescence reporter‐assisted CRISPR/Cas9 gene editing in maize

Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR‐associated protein 9 (Cas9) has revolutionized functional gene analysis and genetic improvement. While reporter‐assisted CRISPR/Cas systems can greatly facilitate the selection of genome‐edited plants produced via stable transformation, this approach has not been well established in seed crops. Here, we established the seed fluorescence reporter (SFR)‐assisted CRISPR/Cas9 systems in maize (Zea mays L.), using the red fluorescent DsRED protein expressed in the endosperm (En‐SFR/Cas9), embryos (Em‐SFR/Cas9), or both tissues (Em/En‐SFR/Cas9). All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out. We describe several case studies of the implementation of En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐ SFR/Cas9 to identify plants not harboring the genome‐editing cassette but carrying the desired mutations at target genes in single genes or in small‐scale mutant libraries, and report on the successful generation of single‐target mutants and/or mutant libraries with En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐SFR/Cas9. SFR‐assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.     

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.     

Base editing with high efficiency in allotetraploid oilseed rape by A3A‐PBE system

CRISPR/Cas‐base editing is an emerging technology that could convert a nucleotide to another type at the target site. In this study, A3A‐PBE system consisting of human A3A cytidine deaminase fused with a Cas9 nickase and uracil glycosylase inhibitor was established and developed in allotetraploid Brassica napus. We designed three sgRNAs to target ALS, RGA and IAA7 genes, respectively. Base‐editing efficiency was demonstrated to be more than 20% for all the three target genes. Target sequencing results revealed that the editing window ranged from C1 to C10 of the PAM sequence. Base‐edited plants of ALS conferred high herbicide resistance, while base‐edited plants of RGA or IAA7 exhibited decreased plant height. All the base editing could be genetically inherited from T₀ to T₁ generation. Several Indel mutations were confirmed at the target sites for all the three sgRNAs. Furthermore, though no C to T substitution was detected at the most potential off‐target sites, large‐scale SNP variations were determined through whole‐genome sequencing between some base‐edited and wild‐type plants. These results revealed that A3A‐PBE base‐editing system could effectively convert C to T substitution with high‐editing efficiency and broadened editing window in oilseed rape. Mutants for ALS, IAA7 and RGA genes could be potentially applied to confer herbicide resistance for weed control or with better plant architecture suitable for mechanic harvesting.     

Reduced fire blight susceptibility in apple cultivars using a high‐efficiency CRISPR/Cas9‐FLP/FRT‐based gene editing system

The bacterium Erwinia amylovora, the causal agent of fire blight disease in apple, triggers its infection through the DspA/E effector which interacts with the apple susceptibility protein MdDIPM4. In this work, MdDIPM4 knockout has been produced in two Malus × domestica susceptible cultivars using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Fifty‐seven transgenic lines were screened to identify CRISPR/Cas9‐induced mutations. An editing efficiency of 75% was obtained. Seven edited lines with a loss‐of‐function mutation were inoculated with the pathogen. Highly significant reduction in susceptibility was observed compared to control plants. Sequencing of five potential off‐target sites revealed no mutation event. Moreover, our construct contained a heat‐shock inducible FLP/FRT recombination system designed specifically to remove the T‐DNA harbouring the expression cassettes for CRISPR/Cas9, the marker gene and the FLP itself. Six plant lines with reduced susceptibility to the pathogen were heat‐treated and screened by real‐time PCR to quantify the exogenous DNA elimination. The T‐DNA removal was further validated by sequencing in one plant line. To our knowledge, this work demonstrates for the first time the development and application of a CRISPR/Cas9‐FLP/FRT gene editing system for the production of edited apple plants carrying a minimal trace of exogenous DNA.     

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.     

CRISPR/Cas9 editing of carotenoid genes in tomato

CRISPR/Cas9 technology is rapidly spreading as genome editing system in crop breeding. The efficacy of CRISPR/Cas9 in tomato was tested on Psy1 and CrtR-b2, two key genes of carotenoid biosynthesis. Carotenoids are plant secondary metabolites that must be present in the diet of higher animals because they exert irreplaceable functions in important physiological processes. Psy1 and CrtR-b2 were chosen because their impairment is easily detectable as a change of fruit or flower color. Two CRISPR/Cas9 constructs were designed to target neighboring sequences on the first exon of each gene. Thirty-four out of forty-nine (69%) transformed plants showed the expected loss-of-function phenotypes due to the editing of both alleles of a locus. However, by including the seven plants edited only at one of the two homologs and showing a normal phenotype, the editing rate reaches the 84%. Although none chimeric phenotype was observed, the cloning of target region amplified fragments revealed that in the 40% of analyzed DNA samples were present more than two alleles. As concerning the type of mutation, it was possible to identify 34 new different alleles across the four transformation experiments. The sequence characterization of the CRISPR/Cas9-induced mutations showed that the most frequent repair errors were the insertion and the deletion of one base. The results of this study prove that the CRISPRCas9 system can be an efficient and quick method for the generation of useful mutations in tomato to be implemented in breeding programs.