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CRISPR-Cas9-Edited Site Sequencing (CRES-Seq): An Efficient and High-Throughput Method for the Selection of CRISPR-Cas9-Edited Clones.
The emergence of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR-Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells). Here we described a next-generation sequencing method called CRISPR-Cas9 Edited Site Sequencing (CRES-Seq) for the efficient and high-throughput screening of CRISPR-Cas9-edited clones. CRES-Seq facilitates the precise genotyping up to 96 CRISPR-Cas9-edited sites (CRES) in a single MiniSeq (Illumina) run with an approximate sequencing cost of $6/clone. CRES-Seq is particularly useful when multiple genes are simultaneously targeted by CRISPR-Cas9, and also for screening of clones generated from multiallelic genes/polyploid cells. © 2018 by John Wiley & Sons, Inc.
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Synthetic molecular sensors based on CRISPR-Cas9 redirect anticancer signal flows to treat retinoblastomas.
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Cas9-NG Greatly Expands the Targeting Scope of the Genome-Editing Toolkit by Recognizing NG and Other Atypical PAMs in Rice.
CRISPR technologies enabling precise genome manipulation are valuable for gene function studies and molecular crop breeding. However, the requirement of a protospacer adjacent motif (PAM), such as NGG and TTN, for Cas protein recognition restricts the selection of targetable genomic loci in practical applications of CRISPR technologies. Recently Cas9-NG, which recognizes a minimal NG PAM, was reported to expand the targeting space of genome editing in human cells, but it remains unclear whether this Cas9 variant can be used in plants. In this study, we evaluated the nuclease activity of Cas9-NG toward various NGN PAMs by targeting endogenous genes in transgenic rice. We found that Cas9-NG edits all NGG, NGA, NGT, and NGC sites with impaired activity, while the gene-edited plants were dominated by monoallelic mutations. Cas9-NG-engineered base editors were then developed and used to generate OsBZR1 gain-of-function plants that can not be created by other available Cas9-engineered base editors. Moreover, we showed that a Cas9-NG-based transcriptional activator efficiently upregulated the expression of endogenous target genes in rice. In addition, we discovered that Cas9-NG recognizes NAC, NTG, NTT, and NCG apart from NG PAM. Together, these findings demonstrate that Cas9-NG can greatly expand the targeting scope of genome-editing tools, showing great potential for targeted genome editing, base editing, and genome regulation in plants.
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Putting CRISPR into African hands to future-proof crops.
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Characteristic and inheritance analysis of targeted mutagenesis mediated by genome editing in rice.
The transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) systems are two current genome editing technologies. Here, we compare and analyze the characteristics of the targeted mutations mediated by these two systems, such as efficiency, type, position, time, and genetic patterns. Both the TALEN and CRISPR/Cas9 systems can induce site-specific mutations in T0 rice plants effectively, but CRISPR/Cas9 is more effective. The major mutation type in both systems is the short insertion/deletion(InDel) mutation within 10 base pairs: deletions ranging from 1 to 10 bps are more often in TALEN, and 1bp insertions are more often in CRISPR/Cas9. Moreover, double-strand breaks (DSBs) generated by CRISPR/Cas9 are more precise than TALEN. In addition, DSBs could be repaired by the homologous recombination at a low frequency, causing DNA fragment duplication mutations. In some cases, the DNA fragments between the two close targets are deleted or inverted, and the mutation efficiency does not positively correlatewith the mutation efficiency of each target. Mutagenesis mediated by the TALEN or CRISPR/Cas9 system can occur as early as in transformed callus cells, and less frequently in somatic cells. Consequently, four different mutation types are formed, including homozygous, heterozygous, bi-allelic and chimeric mutations, with bi-allelic mutations having the highest rate and chimeric mutations having the lowest rate. All, except chimeric mutations, can descend stably into the next generation.
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A Catalogue of Cas9 Orthologs to Advance Genome Engineering.
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CRISPR expands insight into the mechanisms of ALS and FTD.
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[New CRISPR methods and more accurate precision medicine].
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CRISPR therapeutics push into human testing.
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Hijack of CRISPR defences by selfish genes holds clinical promise.
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