Large dataset enables prediction of repair after CRISPR–Cas9 editing in primary T cells

Leenay, Ryan T.; Aghazadeh, Amirali; Hiatt, Joseph; Tse, David; Roth, Theodore L.; Apathy, Ryan; Shifrut, Eric; Hultquist, Judd F.; Krogan, Nevan J.; Wu, Zhenqin; Cirolia, Giana; Canaj, Hera; Leonetti, Manuel D.; Marson, Alexander; May, Andrew P.; Zou, James

doi:10.1038/s41587-019-0203-2

Cited by 96 publications

(117 citation statements)

References 19 publications

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“…We will also assess if the removal of the two gRNA target sites from the donor plasmid would facilitate HDR-based gene insertion in rice. It would also be informative to determine whether certain gRNAs can facilitate large DNA insertions during the DNA-repair process as has been demonstrated for human primary T cells 52 .…”

Section: Discussionmentioning

confidence: 99%

Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9

et al. 2020

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Targeted insertion of transgenes at predetermined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops.

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Section: Discussionmentioning

confidence: 99%

Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9

et al. 2020

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“…Third, indel mutations formed at DSB sites generated by Cas nucleases in CRISPRmediated HDR experiments can result in defective PCR amplification of indel-containing loci that have not undergone HDR and therefore cause an overestimation of the frequency of HDR events by DTECT ( Figures S11A and S11B). However, given that the mutagenic spectrum of indel mutations induced by any sgRNA is predictable (Allen et al, 2019;Leenay et al, 2019;Shen et al, 2018;van Overbeek et al, 2016; inDelphi, https://indelphi.giffordlab.mit.edu/), the negative impact of indel mutations on DTECT-based quantification of CRISPR-mediated HDR events can be avoided by introducing the desired genomic changes in indel-free regions adjacent to CRISPR-induced cut sites ( Figures S11C and S11D). This limitation does not affect the detection of CRISPR-mediated base editing and prime editing events or naturally occurring genetic variants, which are accompanied by either very low frequency (Anzalone et al, 2019;Gaudelli et al, 2017;Komor et al, 2017;Yeh et al, 2018) or absence of DSB-induced indel formation, respectively.…”

Section: Discussionmentioning

confidence: 99%

Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures

et al. 2020

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“…However, the efficiency of conversion of different pGuides varied and appeared to depend upon both the spacer 18 sequence and the stem sequence. Nucleotide composition is known to greatly influence DNA repair outcomes at spCas9 generated DSBs (Allen et al, 2018;Chakrabarti et al, 2019;Leenay et al, 2019;Shen et al, 2018;van Overbeek et al, 2016). It is possible that some stem sequences are less amenable to the desired MMEJ repair event if cryptic microhomologies occur because of the nucleotide composition of the spacer region or between the target site in the stem and the ribozyme DNA sequence.…”

Section: Discussionmentioning

confidence: 99%

Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities

Clarke

Terry

Pennington

et al. 2020

Preprint

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Genetic manipulation of mammalian cells is instrumental to modern biomedical research but is currently limited by poor capabilities of sequentially controlling multiple manipulations in cells. Currently, either highly multiplexed manipulations can be delivered to populations of cells all at one time, or gene regulatory sequences can be engineered to conditionally activate a few manipulations within individual cells. Here, we provide proof-of-principle for a new system enabling multiple genetic manipulations to be executed as a preprogrammed cascade of events. The system leverages the programmability of the S.pyogenes Cas9 RNA-guided nuclease and is based on flexible arrangements of individual modules of activity. The basic module consists of an inactive single guide RNA (sgRNA) -like component that is converted to an active state through the effects of another sgRNA. Modules can be arranged to bring about an algorithmic program of genetic manipulations without the need for engineering cell type specific promoters or gene regulatory sequences. With the expanding diversity of available tools that utilize spCas9 to edit, repress or activate genes, this sgRNA-based system provides multiple levels for interfacing with host cell biology. In addition, ability of the system to progress through multiple modules from episomal plasmid DNA makes it suitable for applications sensitive to the presence of heterologous genomic DNA sequences and broadly applicable to biomedical research and mammalian cell engineering.

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Large dataset enables prediction of repair after CRISPR–Cas9 editing in primary T cells

Cited by 96 publications

References 19 publications

Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9

Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9

Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures

Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities

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