scholarly journals gRNA validation for wheat genome editing with the CRISPR-Cas9 system

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Taj Arndell ◽  
Niharika Sharma ◽  
Peter Langridge ◽  
Ute Baumann ◽  
Nathan S. Watson-Haigh ◽  
...  

Abstract Background The CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate. Results The seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (≥20 bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat. Conclusions We have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.

2019 ◽  
Vol 20 (15) ◽  
pp. 3623 ◽  
Author(s):  
Tobias Bruegmann ◽  
Khira Deecke ◽  
Matthias Fladung

CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.


2021 ◽  
Author(s):  
Jeffrey C Medley ◽  
Shilpa Hebbar ◽  
Joel T Sydzyik ◽  
Anna Y. Zinovyeva

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize off-target sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.


Author(s):  
Anindya Bandyopadhyay ◽  
Nagesh Kancharla ◽  
vivek javalkote ◽  
santanu dasgupta ◽  
Thomas Brutnell

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA (tracrRNA), targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.


mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhiwei Hu ◽  
Yannan Wang ◽  
Qian Liu ◽  
Yan Qiu ◽  
Zhiyu Zhong ◽  
...  

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.


2021 ◽  
Author(s):  
M. Kyle Cromer ◽  
Valentin V. Barsan ◽  
Erich Jaeger ◽  
Mengchi Wang ◽  
Jessica P. Hampton ◽  
...  

As CRISPR-based therapies enter the clinic, evaluation of the safety remains a critical and still active area of study. While whole genome sequencing is an unbiased method for identifying somatic mutations introduced by ex vivo culture and genome editing, this methodology is unable to attain sufficient read depth to detect extremely low frequency events that could result in clonal expansion. As a solution, we utilized an exon capture panel to facilitate ultra-deep sequencing of >500 tumor suppressors and oncogenes most frequently altered in human cancer. We used this panel to investigate whether transient delivery of high-fidelity Cas9 protein targeted to three different loci (using guide RNAs (gRNAs) corresponding to sites at AAVS1, HBB, and ZFPM2) at day 4 and day 10 timepoints post-editing resulted in the introduction or enrichment of oncogenic mutations. In three separate primary human HSPC donors, we identified a mean of 1,488 variants per Cas9 treatment (at <0.07% limit of detection). After filtering to remove germline and/or synonymous changes, a mean of 3.3 variants remained per condition, which were further reduced to six total mutations after removing variants in unedited treatments. Of these, four variants resided at the predicted off-target site in the myelodysplasia-associated EZH2 gene that were subject to ZFPM2 gRNA targeting in Donors 2 and 3 at day 4 and day 10 timepoints. While Donor 1 displayed on-target cleavage at ZFPM2, we found no off-target activity at EZH2. Sanger sequencing revealed a homozygous single nucleotide polymorphism (SNP) at position 14bp distal from the Cas9 protospacer adjacent motif in EZH2 that eliminated any detectable off-target activity. We found no evidence of exonic off-target INDELs with either of the AAVS1 or HBB gRNAs. These findings indicate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP outside the seed region of the gRNA protospacer is sufficient to eliminate Cas9 off-target activity with this method of delivery into primary, repair competent human HSPCs.


2020 ◽  
Vol 6 (6) ◽  
pp. 372-384
Author(s):  
Yating Chen ◽  
Wenwen Mao ◽  
Ting Liu ◽  
Qianqian Feng ◽  
Li Li ◽  
...  

2016 ◽  
Vol 4 (2) ◽  
pp. 75-82 ◽  
Author(s):  
Gaoyuan Song ◽  
Meiling Jia ◽  
Kai Chen ◽  
Xingchen Kong ◽  
Bushra Khattak ◽  
...  
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