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Prew, Michelle

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Prew

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Michelle

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Prew, Michelle

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2015 - 20162

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Author's Publications: search.filters.isAuthorOfPublication.1d06577a-d290-4e66-86b8-3e6b37165c17

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    High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets
    (2015) Kleinstiver, Benjamin; Pattanayak, Vikram; Prew, Michelle; Tsai, Shengdar Q.; Nguyen, Nhu; Zheng, Zongli; Joung, J. Keith
    CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-targets of the broadly used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harboring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Strikingly, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-targets induced by SpCas9-HF1 were not detected. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other RNA-guided nucleases.
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    Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells
    (2016) Kleinstiver, Benjamin; Tsai, Shengdar Q.; Prew, Michelle; Nguyen, Nhu; Welch, Moira M.; Lopez, Jose M.; McCaw, Zack; Aryee, Martin; Joung, J. Keith
    The activities and genome-wide specificities of CRISPR-Cas Cpf1 nucleases1 are not well defined. We show that two Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) have on-target efficiencies in human cells comparable with those of the widely used Streptococcus pyogenes Cas9 (SpCas9)2–5. We also report that four to six bases at the 3’ end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches, but that many of the other bases in this region of the crRNA are highly sensitive to single or double substitutions. Using GUIDE-seq and targeted deep sequencing analyses performed with both Cpf1 nucleases, we were unable to detect off-target cleavage for more than half of 20 different crRNAs. Our results suggest that AsCpf1 and LbCpf1 are highly specific in human cells.