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Engineering CRISPR/LbCas12a for highly efficient, temperature-tolerant plant gene editing.
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CRISPR/Cas9-Induced (CTG⋅CAG)n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing.
Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5' or 3' unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1.
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CRISPR-P 2.0: An Improved CRISPR-Cas9 Tool for Genome Editing in Plants.
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CRISPR genome editing in stem cells turns to gold.
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CRISPR Pigs Portend a New Era of Xenotransplantation.
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Review of Altered inheritance: CRISPR and the ethics of human genome editing by Françoise Baylis.
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A blueprint for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens.
CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation. CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base-editing systems to induce precise cytosine or adenine conversions in P. patens. Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens. These two base editors will facilitate gene functional analysis in P. patens, allowing for site-specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing.
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Multiplex Gene Editing in Rice Using the CRISPR-Cpf1 System.
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Commentary: CRISPR gene editing for inherited retinal dystrophies: Towards clinical translation.
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CRISPR's Twisted Tales: Clarifying Misconceptions about Heritable Genome Editing.
The raging controversy about whether heritable genome editing should be permitted is shaped and structured by the prevailing and countervailing narratives in circulation. In recent years, considerable shortcomings have come to characterize this discourse; it is now time to identify and correct a number of serious misunderstandings and distortions that have taken hold. This essay begins by briefly evaluating reactions to the November 2018 announcement that gene-edited babies had been born; it asserts that widespread agreement about the researcher's recklessness and dire ethical violations concealed deep fault lines among participants in the heritable genome editing debate. It goes on to consider several key omissions and misrepresentations that distort public understanding and undermine genuine debate. It suggests that the conversation must be refocused away from technical, medical, and scientific considerations toward matters of societal meanings, values, context, and consequences. It concludes with criteria for a broadly inclusive and meaningful decision-making process about whether heritable genome editing has any place in the shared and just future to which we aspire.
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