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

Li, Jianying; Manghwar, Hakim; Liang, Sun; Wang, Pengcheng; Wang, Guanying; Sheng, Hanyan; Zhang, Jie; Líu, Hao; Qin, Lei; Rui, Hangping; Li, Bo; Lindsey, Keith; Daniell, Henry; Jin, Shuangxia; Zhang, Xianlong

doi:10.1111/pbi.13020

Cited by 166 publications

(144 citation statements)

References 56 publications

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“…However, it is possible that unintended mutations occur in some somatic cells, rare mutations that can be detected only by using more sensitive methods such as deep sequencing of relevant PCR amplicons. It is also possible that genetic variations from parental plants can alter PAMs and increase the potential off-target effect in more complex and larger polyploid genomes like cotton (Li et al, 2018). Nevertheless, the results indicate that, with careful design of guide RNA and better knowledge of the genome, the CRISPR/ Cas9 system can specifically target the pre-selected sites for mutations.…”

Section: No Off-target Mutation Detected Via Sanger Sequencing Of Pcrmentioning

confidence: 99%

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

Zhang

Hua

Gupta

et al. 2019

Plant Biotechnology Journal

163

104

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Summary 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 T 0 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, Ta CKX 2‐1 , Ta GLW 7 , Ta GW 2, and Ta GW 8 , in T 0 , T 1 , and T 2 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 Ta CKX 2‐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.

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Section: No Off-target Mutation Detected Via Sanger Sequencing Of Pcrmentioning

confidence: 99%

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

Zhang

Hua

Gupta

et al. 2019

Plant Biotechnology Journal

163

104

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“…With the development of modern technologies, plant breeders have the opportunity to overcome this bottleneck. Studies have demonstrated that targeted mutagenesis and precise base editing can be accurately applied for the G. hirsutum genome using genome editing systems [68][69][70][71][72][73][74][75][76][77]. Genome resequencing has identified many domestication genes associated with fiber quality and yield traits.…”

Section: Box 2 a Model For Genetic Breakdown Of Interspecific Hybridmentioning

confidence: 99%

Gossypium Genomics: Trends, Scope, and Utilization for Cotton Improvement

Yang

Qanmber

Wang

et al. 2020

Trends in Plant Science

110

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Cotton (Gossypium spp.) is the most important natural fiber crop worldwide. The diversity of Gossypium species also provides an ideal model for investigating evolution and domestication of polyploids. However, the huge and complex cotton genome hinders genomic research. Technical advances in high-throughput sequencing and bioinformatics analysis have now largely overcome these obstacles, bringing about a new era of cotton genomics. Here, we review recent progress in Gossypium genomics based on whole genome sequencing, resequencing, and comparative genomics, which have provided insights about the genomic basis of fiber biogenesis and the landscape of cotton functional genomics. We address current challenges and present multidisciplinary genomics-enabled breeding strategies covering the breadth of high fiber yield, quality, and environmental resilience for future cotton breeding programs. Gossypium Genomics at a Glance As the world's most important fiber crop and a major source of seed oil and protein, cotton is cultivated in more than 75 countries around the globe [1]. Cotton fibers are seed trichomes, up to 65 mm long, composed of almost pure cellulose, and provide a unique single-celled model system for studying cell elongation and cell wall biogenesis [2]. The Gossypium genus is extraordinarily diverse, with eight diploid genome groups (A-G, and K) and one allopolyploid group (AD) [3,4]. Hybridization and polyploidization of two parental diploids, an A genome-like species with a D genome-like species, has resulted in at least seven allotetraploid species. Two allotetraploid species, Gossypium hirsutum and Gossypium barbadense, evolved independently; these two account for over 90% of annual commercial fiber production. Gossypium is ideal for investigating the origin, evolution, and domestication of polyploids [5-7]. Highlights Cotton is an important natural fiber crop cultivated worldwide that also provides an ideal model for investigating evolution and domestication of polyploids Combinations of the latest technologies, such as optical mapping, high-throughput chromosome conformation capture (Hi-C), and Pacific Biosciences (PacBio) long-reads, have been used to generate multiple high-quality reference genomes of diploid and allotetraploid cotton. Comparative population genomics illuminated the genetic history of cotton domestication and identified the genomic variation determining fiber yield, quality, and stress resistance.

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“…Off-target effects of CRISPR-Cas systems are potential concerns in many of their applications. Whole-genome sequencing (WGS) conducted in Arabidopsis, tomato and cotton showed that off-target activity induced by SpCas9 has rarely occurred (Feng et al 2014;Nekrasov et al 2017;Li et al 2018c;Tang et al 2018). High targeting specificity was also recently reported for Cas12a in rice and cotton Li et al 2019a).…”

Section: Off-target Effects Of Crispr-cas Systemsmentioning

confidence: 94%

CRISPR-Cas nucleases and base editors for plant genome editing

et al. 2019

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Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) and base editors are fundamental tools in plant genome editing. Cas9 from Streptococcus pyogenes (SpCas9), recognizing an NGG protospacer adjacent motif (PAM), is a widely used nuclease for genome editing in living cells. Cas12a nucleases, targeting T-rich PAMs, have also been recently demonstrated in several plant species. Furthermore, multiple Cas9 and Cas12a engineered variants and orthologs, with different PAM recognition sites, editing efficiencies and fidelity, have been explored in plants. These RNA-guided sequence-specific nucleases (SSN) generate double-stranded breaks (DSBs) in DNA, which trigger non-homologous end-joining (NHEJ) repair or homology-directed repair (HDR), resulting in insertion and deletion (indel) mutations or precise gene replacement, respectively. Alternatively, genome editing can be achieved by base editors without introducing DSBs. So far, several base editors have been applied in plants to introduce C-to-T or A-to-G transitions, but they are still undergoing improvement in editing window size, targeting scope, off-target effects in DNA and RNA, product purity and overall activity. Here, we summarize recent progress on the application of Cas nucleases, engineered Cas variants and base editors in plants.

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

Cited by 166 publications

References 56 publications

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

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

Gossypium Genomics: Trends, Scope, and Utilization for Cotton Improvement

CRISPR-Cas nucleases and base editors for plant genome editing

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