Search

MCL-1 is an anti-apoptotic BCL-2 family protein essential for survival of diverse cell types and is a major driver of cancer and chemoresistance. The mechanistic basis for the oncogenic supremacy of MCL-1 among its anti-apoptotic homologs is unclear and implicates physiologic roles of MCL-1 beyond apoptotic suppression. Here we find that MCL-1-dependent hematologic cancer cells specifically rely on fatty acid oxidation (FAO) as a fuel source because of metabolic wiring enforced by MCL-1 itself. We demonstrate that FAO regulation by MCL-1 is independent of its anti-apoptotic activity, based on metabolomic, proteomic, and genomic profiling of MCL-1-dependent leukemia cells lacking an intact apoptotic pathway. Genetic deletion of Mcl-1 results in transcriptional downregulation of FAO pathway proteins such that glucose withdrawal triggers cell death despite apoptotic blockade. Our data reveal that MCL-1 is a master regulator of FAO, rendering MCL-1-driven cancer cells uniquely susceptible to treatment with FAO inhibitors.

CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations at genomic locations that resemble the intended target. These so-called off-target effects can confound research applications and are important considerations for potential therapeutic use. Existing strategies for reducing genome-wide off-targets of the broadly used Streptococcus pyogenes Cas9 (SpCas9) have thus far proven to be imperfect by possessing only partial efficacy and/or other limitations that constrain their use. Here we describe a high-fidelity variant of SpCas9, called SpCas9-HF1, that contains alterations in the amino acid sequence designed to reduce non-specific contacts to the target strand DNA. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of the 37 single-guide RNAs (sgRNAs) tested in human cells. Strikingly, with eight different sgRNAs targeted to standard non-repetitive sequences in human cells, SpCas9-HF1 rendered all or nearly all off-target events imperceptible 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 important and easily employed alternative to wild-type SpCas9 that can eliminate off-target effects when using CRISPR-Cas9 for research and therapeutic applications. Our findings also suggest a general strategy for improving or optimizing the genome-wide specificities of other Cas9 orthologues and engineered variants.

Specificity and targeting range are important considerations when selecting an engineered nuclease platform for functional studies or therapeutic applications. The use of CRISPR/Cas9 to edit virtually any genome has enabled researchers to rapidly knock out genes or to introduce precise genomic changes. A number of studies have explored and improved the specificity of the CRISPR/Cas9 platform but the targeting range remains limited to certain sequences. The most widely used Cas9 from Streptococcus pyogenes (SpCas9) recognizes a protospacer adjacent motif (PAM) of the form NGG, a constraint that restricts the range of sequences that can be edited. Combined with the 5’ nucleotide requirement for guide-RNA expression from a polIII promoter, this restriction can be limiting particularly when one is using CRISPR/Cas9 nucleases to introduce precise mutations by homology-directed repair (HDR), which requires that double-strand breaks be introduced very closely to the site of sequence alteration.To address this challenge, we developed a heterologous genetic system that enabled us to rapidly screen libraries of Cas9 clones and identify variants with distinct cleavage specificities. Using this technology, we identified and optimized two different Cas9 variants that recognize novel PAMs. We demonstrate that these variants enable targeting of sites with non-canonical PAM sequences that were previously inaccessible with wild-type SpCas9, more than doubling the total range of sequences that can be edited by HDR and by error-prone non-homologous end-joining (NHEJ). We demonstrate that these variants can robustly modify endogenous genes in human cells and that they are compatible with previously described specificity improvements to SpCas9 such as truncated gRNAs.Furthermore, we also show Cas9 nucleases from other bacterial species such as Staphylococcus aureus and Streptococcus thermophilus can also be assessed for activity in our heterologous genetic system, suggesting that these alternative Cas9s might also be modified in their PAM specificities using the same approach we undertook with SpCas9. These orthogonal Cas9 nucleases offer the advantage of being substantially smaller in size than SpCas9, an important factor when considering packaging limits for viral delivery of CRISPR/Cas9 components. Taken together, our results more than double the targeting range of CRISPR/Cas9 nuclease platform and improve the prospects for translation of these reagents to clinical settings.