Error‐free recombination in sugarcane mediated by only 30 nucleotides of homology and CRISPR/Cas9 induced DNA breaks or Cre‐recombinase

AbstractScalable isogenic models of cancer-associated mutations are critical to studying dysregulated gene function. Nonsynonymous mutations of splicing factors, which typically affect one allele, are common in many cancers, but paradoxically confer growth disadvantage to cell lines, making their generation and expansion challenging. Here, we combine AAV-intron trap, CRISPR/Cas9, and inducible Cre-recombinase systems to achieve >90% efficiency to introduce the oncogenic K700E mutation in SF3B1, a splicing factor commonly mutated in multiple cancers. The intron-trap design of AAV vector limits editing to one allele. CRISPR/Cas9-induced double stranded DNA breaks direct homologous recombination to the desired genomic locus. Inducible Cre-recombinase allows for the expansion of cells prior to loxp excision and expression of the mutant allele. Importantly, AAV or CRISPR/Cas9 alone results in much lower editing efficiency and the edited cells do not expand due to toxicity of SF3B1-K700E. Our approach can be readily adapted to generate scalable isogenic systems where mutant oncogenes confer a growth disadvantage.

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Author response for "Identification of Retinal Ganglion Cell Types and Brain Nuclei expressing the transcription factor Brn3c/Pou4f3 using a Cre recombinase knock‐in allele"

10.1002/cne.25065/v2/response1 ◽

2020 ◽

Author(s):

Nadia Parmhans ◽

Anne Drury Fuller ◽

Eileen Nguyen ◽

Katherine Chuang ◽

David Swygart ◽

...

Keyword(s):

Transcription Factor ◽

Ganglion Cell ◽

Retinal Ganglion Cell ◽

Cell Types ◽

Cre Recombinase ◽

Author Response ◽

Retinal Ganglion ◽

Brain Nuclei

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Prime-editors (nickases), hRad51–Cas9 nickase fusions and dCas9 have the same problem as conventional CRISPR-Cas9 of plasmid/Cas9 integration after making a double stranded break

10.31219/osf.io/jf6pe ◽

2019 ◽

Author(s):

Sandeep Chakraborty

Keyword(s):

Cellular Response ◽

Sequencing Data ◽

Dna Breaks ◽

Precise Integration ◽

Guide Rna ◽

Double Strand Dna Breaks ◽

Human Therapy ◽

P53 Activation ◽

Programmable Nucleases

‘Prime-editing’ proposes to replace traditional programmable nucleases (CRISPR-Cas9) using a catalytically impaired Cas9 (dCas9) connected to a engineered reverse transcriptase, and a guide RNA encoding both the target site and the desired change. With just a ‘nick’ on one strand, it is hypothe- sized, the negative, uncontrollable effects arising from double-strand DNA breaks (DSBs) - translocations, complex proteins, integrations and p53 activation - will be eliminated. However, sequencing data pro- vided (Accid:PRJNA565979) reveal plasmid integration, indicating that DSBs occur. Also, looking at only 16 off-targets is inadequate to assert that Prime-editing is more precise. Integration of plasmid occurs in all three versions (PE1/2/3). Interestingly, dCas9 which is known to be toxic in E. coli and yeast, is shown to have residual endonuclease activity. This also affects studies that use dCas9, like base- editors and de/methylations systems. Previous work using hRad51–Cas9 nickases also show significant integration in on-targets, as well as off-target integration [1]. Thus, we show that cellular response to nicking involves DSBs, and subsequent plasmid/Cas9 integration. This is an unacceptable outcome for any in vivo application in human therapy.

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