Ivan Tarassov

The CRISPR-Cas12a system, primarily used for nuclear genome editing, operates through RNA-guided cleavage of target DNA. Given the high copy number of mitochondrial DNA (mtDNA) in cells and the pathogenic potential of mtDNA mutations, there is substantial interest in developing methods to selectively eliminate mutant mtDNA without affecting wild-type molecules. We suggest using AsCas12a, with its shorter CRISPR RNA (crRNA) and proven ability to be targeted into human mitochondria, to perform selective cleavage of mutant mtDNA. Here, we assess the potential of AsCas12a and various engineered crRNAs to discriminate between wild-type and mutant mtDNA in vitro and in living human cells. By exploring chemically synthesized full and split crRNAs with various modifications, we identified crRNAs capable of targeting specific mutant mtDNA. We demonstrated the strong influence of magnesium concentration on the ability of crRNAs to discriminate point mutations. Finally, we show that AsCas12a-fused cytosine base editor can be addressed to mitochondria and can direct low but significant specific base editing of mtDNA in human cells. Our study suggests AsCas12a as a promising tool for targeted mutant mtDNA elimination or editing.

Summary: Mutations in the mitochondrial DNA (mtDNA) are associated with severe human diseases, lacking efficient therapies. Direct correction of mtDNA mutations may offer a cure for such diseases. We propose a novel strategy based on double-stranded DNA (dsDNA) oligonucleotide delivery into mitochondria and intrinsic microhomology-mediated end joining (MMEJ) for mtDNA editing. This strategy enables introduction of multiple predefined nucleotide changes in mtDNA. For this, the presence of MMEJ activity in the human mitochondrial lysates was confirmed. 49 bp DNA oligonucleotide duplexes, fused to an RNA hairpin previously identified as a mitochondrial import signal, were delivered into the mitochondria of cultured human cells. Delivery of these donor dsDNA molecules, homological to an ND4 site of mtDNA and bearing designed nucleotide changes, led to a low but statistically significant introduction of the designed nucleotide changes into mtDNA. Donor dsDNA delivery combined with the CRISPR/mito-AsCas12a system also resulted in a statistically significant number of an expected concomitant change of five nucleotides distributed across a 16-nucleotide ND4 site of the mitochondrial genome. The proposed strategy may become an efficient mtDNA editing tool suitable for the correction of near-homoplasmic mutations such as Leber’s Hereditary Optic Neuropathy (LHON)-associated mutations in the ND4 gene of mtDNA.

Mutations in mitochondrial DNA (mtDNA) contribute to various neuromuscular diseases, with severity depending on heteroplasmy level when mutant and wild-type mtDNA coexist within the same cell. Developing methods to model mtDNA dysfunction is crucial for experimental therapies. Here, we adapted the Type V CRISPR–AsCas12a system, which recognizes AT-rich PAM sequences, for targeted editing of human mtDNA. We demonstrated that mitochondrial targeting sequence (MTS) from Neurospora crassa ATPase subunit 9 efficiently addressed the AsCas12a effector nuclease into human mitochondria. When programmed with two CRISPR RNAs (crRNAs) targeting distant regions of mtDNA, the mito-AsCas12a can cleave mtDNA, enabling generation of deletions in cultured human cells. Next generation sequencing of the deletions boundaries confirmed mtDNA ligation after the cleavage by the mitoCRISPR–AsCas12a system. Therefore, we provide experimental data proving that a CRISPR system has potential to be used for precise mtDNA manipulation, offering a promising tool for generating predefined deletions in mtDNA and creating cellular models of mitochondrial disorders.

Mitochondrial endonuclease G (EndoG) contributes to chromosomal degradation when it is released from mitochondria during apoptosis. It is presumed to also have a mitochondrial function because EndoG deficiency causes mitochondrial dysfunction. However, the mechanism by which EndoG regulates mitochondrial function is not known. Fat accumulation in metabolic dysfunction–associated steatotic liver disease (MASLD), which is more common in men, is caused in part by mitochondrial dysfunction. EndoG expression is reduced in MASLD liver, and EndoG deficiency causes MASLD in an obesity-independent manner but only in males. EndoG promotes mitochondrial respiration by resolving mitochondrial tRNA/DNA hybrids formed during mtDNA transcription by recruiting RNA helicase DHX30 to unwind them. EndoG also cleaves off the 3′-end of the H-strand transcript that can prevent mt-tRNA ^Thr precursor cloverleaf-folding, and processing, which increases mt-tRNA ^Thr production and mitochondrial translation. Using fluorescent lifetime imaging microscopy technology to visualize oxygen consumption at the individual mitochondrion level, we found that EndoG deficiency leads to the selective loss of a mitochondrial subpopulation with high-oxygen consumption. This defect was reversed with mt-tRNA ^Thr supplementation. Thus, EndoG promotes mitochondrial respiration by selectively regulating the production of mt-tRNA ^Thr in male mice.

Mutations in mitochondrial DNA (mtDNA) contribute to various neuromuscular diseases, with severity depending on heteroplasmy level when mutant and wild-type mtDNA coexist within the same cell. Developing methods to model mtDNA dysfunction is crucial for experimental therapies. Here, we adapted the Type V CRISPR-AsCas12a system, which recognizes AT-rich PAM sequences, for targeted editing of human mtDNA. We show that AsCas12a effector, fused with a mitochondrial targeting sequence (MTS) from Neurospora crassa ATPase subunit 9, is efficiently addressed into human mitochondria and induces specific mtDNA cleavage in human cells. As a proof-of-concept, we demonstrate that AsCas12a, complexed with two crRNAs targeting distant regions of human mtDNA, introduces specific deletions in mtDNA. For the first time, we provide experimental data proving that a CRISPR system can be used not only for mtDNA degradation but also for precise mtDNA manipulation, offering a potential therapeutic avenue to address mitochondrial disorders.

Transfer RNA dynamics contribute to cancer development through regulation of codon-specific messenger RNA translation. Specific aminoacyl-tRNA synthetases can either promote or suppress tumourigenesis. Here we show that valine aminoacyl-tRNA synthetase (VARS) is a key player in the codon-biased translation reprogramming induced by resistance to targeted (MAPK) therapy in melanoma. The proteome rewiring in patient-derived MAPK therapy-resistant melanoma is biased towards the usage of valine and coincides with the upregulation of valine cognate tRNAs and of VARS expression and activity. Strikingly, VARS knockdown re-sensitizes MAPK-therapy-resistant patient-derived melanoma in vitro and in vivo. Mechanistically, VARS regulates the messenger RNA translation of valine-enriched transcripts, among which hydroxyacyl-CoA dehydrogenase mRNA encodes for a key enzyme in fatty acid oxidation. Resistant melanoma cultures rely on fatty acid oxidation and hydroxyacyl-CoA dehydrogenase for their survival upon MAPK treatment. Together, our data demonstrate that VARS may represent an attractive therapeutic target for the treatment of therapy-resistant melanoma.

Proper RNA localisation is essential for physiological gene expression. Various kinds of genome-wide approaches permit to comprehensively profile subcellular transcriptomes. Among them, cell fractionation methods, that couple RNase treatment of isolated organelles to the sequencing of protected transcripts, remain most widely used, mainly because they do not require genetic modification of the studied system and can be easily implemented in any cells or tissues, including in non-model species. However, they suffer from numerous false-positives since incompletely digested contaminant RNAs can still be captured and erroneously identified as resident transcripts. Here we introduce Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq) as a new subcellular transcriptomics approach that efficiently bypasses this caveat. CoLoC-seq leverages classical enzymatic kinetics and tracks the depletion dynamics of transcripts in a gradient of an exogenously added RNase, with or without organellar membranes. By means of straightforward mathematical modelling, CoLoC-seq infers the localisation topology of RNAs and robustly distinguishes between genuinely resident, luminal transcripts and merely abundant surface-attached contaminants. Our generic approach performed well on human mitochondria and is in principle applicable to other membrane-bounded organelles, including plastids, compartments of the vacuolar system, extracellular vesicles, and viral particles.

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5′-scaffold moiety and variable 3′-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.