A quick guide to CRISPR sgRNA design tools

Brazelton, Antonio; Zarecor, Scott; Wright, David; Wang, Yuan; Liu, Jie; Chen, Keting; Yang, Bing; Lawrence‐Dill, Carolyn J.

doi:10.1080/21645698.2015.1137690

Cited by 85 publications

(47 citation statements)

References 48 publications

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“…To evaluate off‐target activities of Cas9 and Cas12a in maize, we first examined how specific our intended on‐target sites were relative to the maize genome. To do this, we checked for on‐ and off‐target sites for B73 and B104 using the CRISPR Genome Analysis Tool (CGAT; Brazelton et al ., ), and confirmed that our gRNAs and crRNAs have unique targeting sequence within the maize genome. Next we performed a more in‐depth in silico identification of closely related sites (off‐targets) using Cas‐OFFinder (Bae et al ., ).…”

Section: Resultssupporting

confidence: 84%

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

Lee

Zhang

Kleinstiver

et al. 2018

Plant Biotechnology Journal

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196

151

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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.

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Section: Resultssupporting

confidence: 84%

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

Lee

Zhang

Kleinstiver

et al. 2018

Plant Biotechnology Journal

Self Cite

196

151

View full text Add to dashboard Cite

show abstract

“…The guide RNA spacer sequences were designed based on the maize B73 reference genome sequence (Schnable et al, 2009) using the CRISPR Genome Analysis Tool (Brazelton et al, 2015; http://cbc.gdcb.iastate.edu/cgat/). The relevant target regions in Hi-II and B104 genotypes were PCR-amplified and confirmed by sequencing.…”

Section: Targeted Mutagenesis Strategymentioning

confidence: 99%

An Agrobacterium‐delivered CRISPR/Cas9 system for high‐frequency targeted mutagenesis in maize

Char

Neelakandan

Nahampun

et al. 2016

Plant Biotechnology Journal

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307

172

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Summary CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease‐based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium‐delivered CRISPR/Cas9 for high‐frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4‐reductase or anthocyaninless genes (a1 and a4). T0 transgenic events carrying mono‐ or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi‐II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target‐specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.

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“…Although multiple CRISPR sequence design tools exist, many software programs fully rely on computational analysis, resulting in the design of numerous sgRNAs with no mechanism to sort them (Brazelton et al, 2016). In addition, PRV genomes contain high GC content.…”

Section: Discussionmentioning

confidence: 99%

Live attenuated pseudorabies virus developed using the CRISPR/Cas9 system

et al. 2016

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Currently, pseudorabies virus (PRV) variant strains are outbreaking in China; these variants belong to genotype II PRV. The traditional Bartha-K61 vaccine has failed to provide complete protection against the emergent variant strains. Therefore, rapid attenuation of current epidemic strains is needed for effective PRV control. In this study, we report a rapid method for editing the PRV genome using the CRISPR-Cas9 system. We developed a triple gE/gI/TK gene-inactivated HeN1 PRV strain, because mice were more susceptible to PRV infection, we then evaluated the attenuation of PRV in the mice and demonstrated that modified PRV was fully attenuated. Furthermore, the attenuated strain also induced immune protection in response to a parental PRV challenge. Overall, we showed that PRVs can be rapidly attenuated using CRISPR-Cas9 technology, which will be critical for PRV control, especially when new variant PRV strains emerge.

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A quick guide to CRISPR sgRNA design tools

Cited by 85 publications

References 48 publications

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

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

An Agrobacterium‐delivered CRISPR/Cas9 system for high‐frequency targeted mutagenesis in maize

Live attenuated pseudorabies virus developed using the CRISPR/Cas9 system

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