In Vitro Enzymology of Cas9

Anders, Carolin; Jinek, Martin

doi:10.1016/b978-0-12-801185-0.00001-5

Cited by 113 publications

(97 citation statements)

References 49 publications

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“…Escherichia coli strain Rosetta 2 DE3 carrying plasmid pMJ806 was obtained from Addgene. The Cas9 protein was prepared following the protocol as described (Anders and Jinek, ). For in vitro transcription of guide RNA, pT7‐sgRNA derived gRNA‐specific construct was linearized at a unique restriction enzyme site Nco I, followed by dephosphorylation with Shrimp Alkaline Phosphatase (rSAP) (New England Biolabs, Ipswich, MA), and then purified with DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA).…”

Section: Methodsmentioning

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: Methodsmentioning

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

View full text Add to dashboard Cite

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“…Recombinant S. pyogenes Cas9 protein used in this study expresses a C-terminal HA tag and two nuclear localization signal (NLS) peptides that facilitate transport across the nuclear membrane. The protein was expressed and purified as described (Anders and Jinek, 2014) and obtained from the QB3 Macrolab, University of California, Berkeley. Purified Cas9 protein was stored in 20 mM HEPES at pH 7.5 plus 150mM potassium chloride, 10% glycerol, and 1mM tris(2-carboxyethyl)phosphine (TCEP) at −80C.…”

Section: Methodsmentioning

confidence: 99%

A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells

et al. 2016

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SUMMARY New genetic tools are needed to understand the functional interactions between HIV and human host factors in primary cells. We recently developed a method to edit the genome of primary CD4+ T cells by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs). Here, we adapted this methodology to a high-throughput platform for the efficient, arrayed editing of candidate host factors. CXCR4 or CCR5 knock-out cells generated with this method are resistant to HIV infection in a tropism-dependent manner, whereas knock-out of LEDGF or TNPO3 results in a tropism-independent reduction in infection. CRISPR/Cas9 RNPs can furthermore edit multiple genes simultaneously, enabling studies of interactions among multiple host and viral factors. Finally, in an arrayed screen of 45 genes associated with HIV integrase, we identified several candidate dependency/restriction factors, demonstrating the power of this approach as a discovery platform. This technology should accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection.

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“…Cleavage of plasmid or PCR substrates is monitored by agarose gel electrophoresis with an intercalating dye (Figure 3). The reaction rate can strongly vary in function of DNA source and length (PCR product versus plasmid, circular plasmid versus linear plasmid), optimal enzyme and substrate concentrations, and also reaction time points need to be determined empirically [50]. This in vitro test validates sgRNA intrinsic capacity to form cleavage-competent complexes; however, it does not guarantee in vivo effectiveness, which also greatly depends on chromatin accessibility, as previously mentioned.…”

Section: Guide Rna Efficiency and Specificitymentioning

confidence: 98%

Guidelines for Optimized Gene Knockout Using CRISPR/Cas9

et al. 2019

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CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Despite its apparent simplicity, the outcome of a genome-editing experiment can be substantially impacted by technical parameters and biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome-editing experiments.

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In Vitro Enzymology of Cas9

Cited by 113 publications

References 49 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

A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells

Guidelines for Optimized Gene Knockout Using CRISPR/Cas9

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