Billon's lab Journal Club

We highlight a selection of recent articles on Genome Editing and Genome Stability. We are particularly interested in breakthrough articles describing new technologies or new cellular mechanisms.

 

We aim to present one paper each day. Come back tomorrow for another interesting paper on our favorite topics!

A note to readers: We recommend reading the page on a web browser with a computer, not on mobile devices.

January 18th, 2021

The Card1 nuclease provides defence during type-III CRISPR immunity

Type-III CRISPR-Cas systems have the most complex mechanisms of action to provide immunity against invaders. Type-III CRISPR loci contain enzymes exhibiting both CARF (CRISPR-Cas Associated Rossmann Fold) domain, such as the important Csm6/Csx1 nuclease, and domains with regulatory or catalytic functions. Upon recognition of foreign invader's transcripts by Cas10/gRNAs effector complex, the targeted transcript is cleaved resulting in the activation of the ribonuclease Csm6. Csm6 is activated by 3'-5' cyclic oligoadenylate (cA) that bind its CARF domain, generating robust degradation of all foreign RNA transcripts. The presence of additional proteins with a CARF and catalytic domains in Type-III CRISPR loci raises questions on the possibility that other mechanisms of defense exist.

In this paper, Rostol, Xie et al., from the Luciano Marraffini lab investigate the function of a protein which contains both a CARF and an endonuclease-like domain, and which is associated with CRISPR loci. The authors show that this small protein (43.9kDa) degrades ssDNA and ssRNA upstream  of T(A/G) sites in vitro upon activation by cA4, demonstrating that Card1 (cA-activated ssRNAse and ssDNAse 1) is a nuclease against ssRNA and ssDNA, but unlike Csm6 which only targets ssRNA. Next, the authors solved crystal structures of Card1 in different states, enabling atomic resolution of the protein. In particular, the crystal structures revealed that Card1 is a homodimer, the position where cA4 binds between the two domains and a possible mechanism of ssDNA/ssRNA cleavage. Thus, the authors tested the functional role of Card1 in immunity against plasmids and phages. They observed that the ssDNA activity (target plasmid was degraded) but not the ssRNA activity (transcriptome unchanged)​ is involved in immunity against plasmids in a Cas10-dependent manner. The authors propose that Card1 may introduce DNA lesions in the bacterial chromosome to induce cell dormancy. Finally, the authors confirm that Card1 has a role in anti-phage immunity in S.aureus through its RNA activity when it is programmed to target late phage transcripts, similar to Csm6.

This interesting work reveals the diversity of nucleases and associated activities involved in the war between phages and bacteria and opens the door to many more interesting mechanisms.

Extended reads:

Evolutionary and functional classification of the CARF domain superfamily, key sensors in prokaryotic antivirus defense published in NAR

A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems published in Science

Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers published in Nature

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January 16th, 2021

 

Targeted Genome Editing of Bacteria Within Microbial Communities

Micro-organisms relevant for the environment, industries and human health live within communities. The manipulation of the vast majority of microbes is difficult, if not impossible, because the extraction of particular species and their genetic manipulation within a population is hard to achieve. 

In this manuscript, Rubin, Diamond, Cress et al., from the Jillian Banfield and Jennifer Doudna's labs develop two strategies 1) to identify genetically tractable species, and 2) to edit the genome of specific micro-organisms within microbial communities without prior isolation of the targeted bacteria of interest.

The first technology described is called ET-Seq (Environmental Transformation Sequencing). ET-Seq relies on the delivery of mariner transposons which randomly insert into the genome of the micro-organisms. Sequencing of the insertion location and of the whole population reveals which members can be targeted and their proportions within the community. These experiments identify which members are genetically tractable within a bacterial community. The authors tested ET-Seq on a bioreactor, which contains hundreds of different bacteria and were able to edit and isolate certain species that were never isolated before.

The second technology utilizes CRISPR-Cas Tn7 transposases for site-specific editing of selected species within a community of bacteria. First, the authors compared the efficiency and specificity of the two CRISPR-transposon systems, and selected the VcDART system (aka INTEGRATE) because of its high efficiency and specificity. By programming gRNAs specific to the genome of particular bacteria, the authors were able to insert DNA cargos specifically in the genome of the targeted bacteria within the community. In particular as a proof-of-principle, they inserted a DNA sequence to disrupt the pyrF gene, which can be selected with a drug when inactivated. These experiments enable strong enrichment of the selected bacteria generating pure cultures from a mixture of bacteria (See Figure).

Given the importance of the microbiomes for human health, industries and environment, the development of strategies enabling genetic manipulation of selected bacteria within microbiomes represent formidable scientific advances.

Extended reads:

Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration published in Nature.

RNA-guided DNA insertion with CRISPR-associated transposases published in Science.

CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering published in Nature Biotechnology

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January 15th, 2021

 

CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering

 

Site-specific insertion of DNA sequence​s has many genome engineering applications in biotechnology and medicine. The INTEGRATE (Insertion of transposable elements by guide RNA-assisted targeting) system utilizes a Tn7-like transposon from Vibrio cholerae to achieve site-specific DNA integration at gRNA-specified genomic locations, without the need for host factors or DNA repair.

In this paper, Vo et al., from the Sternberg's lab improve the first generation INTEGRATE system that they established in 2019. Here, they built a single-plasmid construct which encodes the elements required to facilitate high efficiency and high specificity integrations. Interestingly, they show that growing bacteria at 30C, instead of 37C, increases the time window for editing enabling high efficiency integration of large DNA cargo (10kb), which are usually hard to integrate. They also develop new INTEGRATE systems using orthologous enzymes to achieve multiplexed integrations of different DNA cargos at various genomic sites. Using this orthologous approach, the authors demonstrate that the combined action of orthologous integrases and recombinases induces programmable deletions. Indeed, the insertion of two adjacent LoxP sites, using RNA-guided orthologous integrases, enable the excision of the genomic sequence located between the 2 loxP sites. The authors also validate the high efficiency and precision of the improved INTEGRATE systems for the engineering of relevant bacterial species, such as Pseudomonas putida (used for industrial applications for its versatile metabolism) and Klebsiella Oxytoca (an emerging pathogen that causes human clinical diseases). Finally, they use INTEGRATE to manipulate specific bacterial species within a complex population of bacteria derived from the mouse gut by targeting a locus that is specific to the intended bacteria.

This impressive work enhances the high versatility and enables novel applications of programmable CRISPR-transposons.

Extended reads:

Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration published in Nature.

RNA-guided DNA insertion with CRISPR-associated transposases published in Science.

Targeted Genome Editing of Bacteria Within Microbial Communities published in BioRxiv.

January 13th, 2021

 

CRISPR-CasΦ from huge phages is a hypercompact genome editor

CRISPR was discovered as a prokaryotic adaptive immune system to fight bacteriophage infection. Surprisingly, CRISPR-Cas systems have recently been identified in the genome of huge bacteriophages. This is a strategy used by phages to eliminate competing phages and gain advantage to propagate.

 

In this study, Pausch, Al-Shayed et al., from the Jennifer Doudna's lab describe the biochemical analysis of a minimal CRISPR system, called CasΦ or Cas12j, derived from huge bacteriophages. The authors show that CasΦ is a single protein of ~70kDa, which corresponds to half the size of Cas9 and Cas12a! The authors also convincingly show that three CasΦ orthologues provide immunity against plasmid transformation suggesting that these systems are functional. They also reveal five important features of CasΦ, which include the ability to process their own crRNAs, require a minimal T-rich PAM (for instance, CasΦ-2 requires a minimal 5'-TB-3' PAM where B is C, G or T) sequence, that the gRNA must be 14 to 20nt long to mediate DNA cleavage, that CasΦ generates staggered 5' overhangs of 8 to 12 nucleotides and that CasΦ exhibits a collateral activity against ssDNA upon recognition of specific DNA target by the sgRNA. The latter feature is important as it could be used for CRISPR diagnostic applications (see other reviews below). Finally and importantly, the authors tested whether CasΦ can be harnessed for genome editing. They delivered CasΦ and gRNA programmed to target and inactivate EGFP in HEK293 cells (See figure, left), as well as to edit plants by the delivery of CasΦ/gRNA ribonucleoprotein complexes. They report a ~30% editing in human cells (mostly deletions) (See figure, right), but very low editing efficiency in plants. 

CasΦ has many important characteristics that makes it a unique CRISPR enzyme: CasΦ not only has a very small size and processes its own crRNA array, but can also be harnessed for genome editing and -probably- for CRISPR diagnostics. Given that most CRISPR applications suffer from the large size of Cas9/Cas12a enzymes for delivery, the discovery and biochemical characterisation of such a small-size and active system is a fabulous addition to the CRISPR toolbox. CasΦ: remember this name!

Extended reads:

Clades of huge phages from across Earth’s ecosystems published in Nature

Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants published in Nature Review Microbiology

Programmed DNA destruction by miniature CRISPR-Cas14 enzymes published in Science

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January 12th, 2021

 

Precise genomic deletions using paired prime editing

 

Precise genomic deletions can be generated by paired CRISPR gRNAs (See Figure a). However, double-strand breaks are repaired by error-prone NHEJ that introduces insertion and deletions (indel) mutations at the junction, thereby limiting the generation of precise deletions. Alternatively, regular prime editing can be utilized to create precise deletions with a unique pegRNA in addition to a gRNA that introduces a nick on the opposing strand (PE3 system) (See Figure b). However, nicks produce a certain frequency of indel mutations limiting the generation of precise deletions with prime editing.

In this elegant paper, Choi et al., from the Jay Shendure's lab develop PRIME-Del a prime editing-based method that introduces precise deletions. This approach uses a paired pegRNAs that are oriented towards one another and which target the opposite DNA strands (See Figure c). The pegRNAs are programmable not only to specify the exact position of the deletion but can also be used to insert additional nucleotides. Here, the authors establish and validate PRIME-Del as a precise deletion method associated with low level of indel mutations. The authors targeted several regions of plasmids or genomic DNA to introduce various programmed deletions of various lengths (up to ~700bp deletion). They also show remarkable example of concomitant introduction of precise deletions and insertions at predicted locations. Finally, the authors show that extended exposure to prime editing reagents increase the level of precision prime editing. While this is not totally surprising, strategies increasing editing levels are important.

Although PRIME-Del has not been tested in clinically relevant systems nor for the generation of precise chromosomal rearrangements, it represents a very promising approach to introduce programmable deletions. The generation of programmed deletions (e.g., repetitive sequences, enhancers, exons...) in high-throughput settings will unlock many important biological questions, and enable several applications for gene therapies. 

Extended reads:

Search-and-replace genome editing without double-strand breaks or donor DNA published in Nature.

CRISPR/Cas9-Mediated Scanning for Regulatory Elements Required for HPRT1 Expression via Thousands of Large, Programmed Genomic Deletions published in American Journal of Human Genetics.

A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells published in Nature Methods.

Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library published in Nature Biotechnology.

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January 11th, 2021

 

Prime editing enables precise genome editing in mouse liver and retina

Prime editing (PE2) is a promising new precision genome editing approach. It utilises prime editing guide RNAs (pegRNA) and a nickase Cas9 to program precise edits introduced by a reverse transcriptase (RT). The RT copies the information encoded into the pegRNA directly into the chromosome achieving programmable precision genome editing. A more efficient approach (called PE3 or PE3b) requires an additional gRNA to bias repair by introducing a nick in the non edited strand. Although prime editing works will modest efficiency in cellular models, it has not been established whether prime editing is efficient and precise in adult animals.

In this manuscript, Jang et al., from the Hyongbum Henry Kim's lab use prime editing to correct a pathogenic mutation in a mouse model of hereditary tyrosinemia and edit the retina of adult mice. Hereditary tyrosinemia type I is caused by mutations in the fumarylacetoacetate hydrolase (FAH) gene. To identify pegRNAs that efficiently correct the targeted FAH mutation, the authors constructed a PE2-NG version, which provides a more flexible PAM sequence, to have a higher targeting range. Using a library of pegRNAs the authors identified the pegRNAs with the highest efficiency (~15-20%) to correct the mutation. Next, the authors delivered plasmids encoding pegRNA and PE2 or PE3 (+sgRNA for PE3) into the liver of adult mice by hydrodynamic injection. 40 or 60 days after injection, 4% (PE2)/11.5% (PE3) of edits were present in the liver and 33%/60% of the cells re-expressed FAH, confirming that prime editors edit the liver of adult mice, thereby correcting disease mutation. This modest editing level was sufficient to partially restore disease phenotypes (splicing defects, weight loss...). As expected from other studies in cellular models, prime editing is precise with low level of indel formation and no bystander mutations, unlike CRISPR-HDR and base editing respectively.

Next, the authors tested whether prime editing can be used in a different tissue (retina) and delivered the prime editing components with viral vectors. In particular, the authors used trans-splicing AAVs to deliver PE2 and pegRNA targeting the ATP7B gene. The trans-splicing AAV system uses two AAV that encode for half of PE2 and reconstitute a functional prime editor in the cells. The authors edited the retina by intravitreal injection of mice. Six weeks after injection, editing level reached ~1.85% without detectable off-target edits at predicted Cas9 off-target sites. Even though there is a very low level of editing, these experiments demonstrate that prime editing edits the retina of adult mice.

This interesting paper shows that prime editing can edit tissues in adult mice without on-target indels and bystander edits, and off-target editing. However, this study also confirm that the development of strategies to improve prime editing efficiencies is important to achieve higher level of precision genome editing.

Extended reads:

Search-and-replace genome editing without double-strand breaks or donor DNA published in Nature.

 

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January 10th, 2021

 

A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression

 

CRISPR-Cas9 is an adaptive immune system guided by two non-coding RNAs called tracrRNA (which helps form the active CRISPR-Cas9 complex) and crRNA (which specifies the target sequence). The fusion of these two RNA molecules form the sgRNA (single guide RNA) that is utilized for genome editing and targeting. The tracrRNA is transcribed from two promoters producing two non-coding elements: a long transcript with an extended 5' end (called tracr-L) (See figure top) and a short form (called tracr-S or tracr-P after processing) (See figure bottom). While the short form is know to mediate CRISPR interference by binding to crRNAs, the biological function of the tracr-L is a complete mistery.

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In this interesting paper, Workman and Pammi et al., from the Joshua Modell's lab use an unbiased genetic screen with transposons to identify elements that restrict bacterial immunity against phages. Surprisingly, they identified the CRISPR locus as the top hit. This result is counter-intuitive given that CRISPR locus encodes for the endonuclease Cas9 and the CRISPR array of crRNAs which program Cas9 against phage genomes. However, this result suggests that certain elements encoded into the CRISPR locus inhibit CRISPR immunity. In particular, the authors convincingly identify and validate the mysterious tracr-L element as a natural inhibitor. They report that in absence of tracr-L the level of the CRISPR components, including Cas9 protein level, are dramatically enhanced suggesting that tracr-L disrupts the expression of the CRISPR locus. They further demonstrate that Cas9 and tracr-L form a sufficient repressor complex. The authors not only reveal that CRISPR and tracr-L bind to the CRISPR promoter, but also that the binding occurs through a 11 nt sequence located on the 5'-end of the tracr-L that matches with the transcription start site of the promoter (see Figure top), thereby repressing transcription of the CRISPR promoter (P cas in Figure). Therefore, tracr-L is a natural single guide RNA that regulates the entire CRISPR-Cas system through the controlled expression of the CRISPR locus. Additionally, tracr-L competes with tracr-S for base pairing with crRNAs, thereby generating inactive gRNAs. This creates a negative feedback control to maintain CRISPR in an inactive state to avoid autoimmunity against the bacterial chromosomal while keeping the surveillance function at low level but operational in case of phage infection.

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Genome editing using CRISPR-Cas9 was quickly democratized because Jennifer Doudna and Emmanuelle Charpentier's labs fused tracr-P to crRNAs to create a single-guide RNA (originally called sgRNA) to enable programmable genome editing with a unique gRNA. With this study we now understand why bacteria have two independent non-coding RNAs instead of one: Bacteria can reconstitute an active CRISPR system while self-controlling its activity by decreasing Cas9 expression and keeping crRNA inactive. This control prevents auto-immunity and facilitates horizontal transfer into new hosts. Brilliant!

January 9th, 2021

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From the News and Views associated with the article.

In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice

 

CRISPR-mediated base editing has the potential to ​correct mutations causing incurable human genetic disorders. Adenine Base Editors (ABE) convert genomic A:T base pairs into G:C by deaminating targeted adenine(s) in cell lines, post-mitotic cells and model organisms. Hutchinson-Gilford progeria syndrome is an autosomal dominant condition caused by a single base change in the genome of the affected individuals. A major problem associated with this disease is that it affects various organs, thereby complicating the development of effective treatments.

Although clinical translation of this work into humans will require many years of work and close monitoring/improvement of AAV due to potential adverse effects (5/9 AAV9-treated mice developed liver cancer), this impressive work provides hope for children living with this devastating disease characterised by a short lifespan (~14 years in humans). Indeed, LMNA c.1824C>T is the most prevalent mutation in humans with progeria. This paper is a beautiful demonstration and confirmation of the strong potential of base editing for the treatment of genetic disorders.

Extended reads:

Programmable base editing of A:T to G:C in genomic DNA without DNA cleavage by Gaudelli et al., from the David Liu's lab at Harvard University published in Nature.

Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction by Koblan et al., from the David Liu's lab at Harvard University published in Nature Biotechnology.

Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity by Richter et al., from the David Liu's lab at Harvard University published in Nature Biotechnology.

Directed evolution of adenine base editors with increased activity and therapeutic application by Gaudelli et al., from Beam Therapeutics published in Nature Biotechnology

In this paper, Koblan, Erbos et al., from the Francis Collins, Jonathan Brown and David Liu's labs use the ABE enzyme to correct the causative mutation of progeria in primary cells derived from patients and in an humanized mice model (See Figure). In particular, the authors used ABEmax-VRQR which encodes an optimized ABE enzyme and which recognizes NGA sequence as a protospacer adjacent motif (PAM), allowing the positioning of the progeria point mutation at an ideal position for targeting and correction. Using a lentivirus to deliver ABE, the authors report an impressive ~90% correction of the LMNA c.1824C>T pathogenic mutation with only ~1-2% bystander mutation -which creates a V690A mutation- and background level of indel formation. Additionally, they confirmed that the correction of the pathogenic mutation rescues the molecular and phenotypic consequences of the cells (e.g., abnormal nucleus morphology, reduction of mis-splicing, reduced progerin level and increased lamin A abundance). Finally, the authors convincingly showed that no DNA or RNA-off target editing was detected in the corrected cells. 

Given these impressive results in primary human cells, the authors tested the possibility of correcting a mouse model of progeria which encodes the complete human LMNA gene (including the c.1824C>T mutation). To deliver ABE, the authors used a systemic delivery of AAV9 (for its broad tissue tropism) with the trans-splicing intein system. This clever system utilizes two AAVs each encoding half ABE. ABE is reconstituted into a full length active enzyme by the action of intein fused to each half of ABE. Humanized mouse model for progeria were treated by a single injection of AAV postnatally at day 14. This treatment resulted in 10 to 60% correction of the mutation in various tissues after few months. Despite a modest editing level in the aorta, the authors show that the treatment improves vascular pathology, which is known to cause morbidity and mortality in human patients. More importantly, the median lifespan of the treated mice increased by 2.4 fold. This is an impressive result given that the mice received a single injection of the treatment!

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January 8th, 2021

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CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia

 

Mutations in the hemoglobin-beta subunit gene (HBB) cause the most common monogenic inherited human diseases: Beta-thalasemia and Sickle Cell Disease. BCL11A is a transcription factor that represses fetal hemoglobin in erythroid cells (Figure, panel A)BCL11A expression is influenced by an erythroid-specific enhancer which is controlled by the GATA1 transcription factor. Observations suggest that patients with persistant expression of the fetal hemoglobin after birth have a lower disease severity, suggesting that disruption of GATA1 binding site could induce a reduction in BCL11A expression and potentially restore disease phenotypes by restoring expression of fetal hemoglobin.

 

In this paper, the authors disrupt GATA1 binding site within the BCL11A enhancer with CRISPR (Figure, panel B) in CD34+ hematopoietic stem and progenitor cells. This is a clever approach given that double-strand break repair by NHEJ is more frequent that the error-free HR. Using this approach, the authors obtained ~80% editing frequency, thereby increasing fetal hemoglobin level. Cells successfully engrafted in immunocompromised mice suggesting that editing does not negatively affect cell engraftment. Two patients, one with Beta-thalasemia and one with Sickle Cell Disease, were enrolled to test such approach. Their CD34+ hematopoietic stem and progenitor cells were collected and edited. Patient 1 had 68.9% editing efficiency and Patient 2 had ~80% editing. The authors confirmed that after transfusion of the edited cells, the cells persisted and that fetal hemoglobin level increased rapidly. Level of fetal hemoglobin was sustained throughout the >1 year follow-up.

Although the two patients had many adverse effects, including a handful of serious effects, all effects were resolved with treatments. The exciting results of this study are a complete elimination of vaso-occlusive episodes or need for transfusion for these patients. This is an exceptional improvement for their quality of life given that before treatment patient 1 had received 34 transfusions per year since birth, and patient 2 had suffered on average 7 severe vaso-occlusive episodes, 3.5 hospitalisations and 5 transfusions each year. The use of CRISPR to cure the most frequent inherited genetic disorders in humans is on.

Extended reads:

Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype published in JCI.

An Erythroid Enhancer of BCL11A Subject to Genetic Variation Determines Fetal Hemoglobin Level published in Science.

BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis published in Nature.

Highly efficient therapeutic gene editing of human hematopoietic stem cells published in Nature Medicine

  

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January 7th, 2021

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FAM72A antagonizes UNG2 to promote mutagenic uracil repair during antibody maturation

AID deaminates cytidine to initiate class-switch recombination (CSR) and hypersomatic mutation (HSM) at immunoglobulin genes in B cells. Cytidine deamination creates uracils which induces U:G mispairs. The Base Excision Repair (BER) through the uracil-N glycosylases 2 (UNG2) enzyme repairs uracils to restaure the genomic sequence. In addition, the Mismatch Repair (MMR) pathway is also utilized to promote CSR and create mutations at the immunoglobulin locus.

In this paper, Feng et al. from the Alberto Martins's lab at The University of Toronto utilize unbiased CRISPR screens to identify novel genes involved in CSR. The authors identified the UNG2-interactor FAM72A as a novel factor for which depletion robustly decreases CSR from IgM to IgA in a model system. Importantly, FAM72A expression is increased in B cells upon LPS stimulation as well as in germinal centers. Remarkably, the authors observed that loss of FAM72A is defective in CSR in primary B cells confirming the critical importance of FAM72A for CSR in vivo. By studying the mutagenesis profile of mutant cells in germinal centers, the lymphoid site where B cells induce CSR and HSM upon antigen activation, the authors found that FAM72A depletion causes a reduction in mutation frequency. Additionally, by employing genetic interaction studies by inactivating genes, the authors showed that FAM72A, UNG (UNG encodes for both nuclear UNG2 and mitochondrial UNG1) and MSH2 (MMR gene) are epistatic for CSR. The authors then hypothesized that FAM72A inhibits UNG2 through their direct interaction. While the authors showed a rather small effect of FAM72A on UNG2 activity using recombinant proteins in vitro, they show a drastic increase of UNG2 protein level in the absence of FAM72A suggesting that FAM72A might antagonize UNG2 by affecting its stability (it is not demonstrated if it's through their direct interaction).

This interesting paper reveals a novel regulatory mechanism that controls UNG2 activity and impacts antibody maturation in B cells.

Extended reads:

AID in Antibody Diversification: There and Back Again by Feng et al., from the Alberto Martin's lab at the University of Toronto published in Trends in Immunology.

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Tn7 transposons have co-opted natural CRISPR-Cas systems to mediate gRNA-dependent transposition and self-propagate within bacterial populations.

In this paper, Petassi et al. from the Joseph Peters lab at Cornell University present a rigorous bioinformatic analysis of various elements of CRISPR loci associated with Tn7-like transposons. The authors confirmed that all the integration events identified in bacterial genomes are mediated by gRNAs encoded within the associated CRISPR arrays. While CRISPR systems have evolved to target and destroy foreign DNA, transposons self-target the host chromosomal DNA. The authors described multiple mechanisms that allow transposition within the bacterial chromosome, but block self-targeting by CRISPR nucleases which would otherwise induce cell death. Among the identified mechanisms, the presence of mismatches in spacers, the presence of atypical gRNA structures and specific transcriptional regulations. 

In 2019, CRISPR-transposons have been repurposed as programmable tools for site-specific integration of a large variety of DNA sequences, without introducing double-strand breaks. The mechanisms for gRNA privatization, identified in this study, will undoubtedly be useful for higher control of genome engineering applications mediated by RNA-guided integrases.

Extended reads:

Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration by Klompe et a., from the Sam Sternberg's lab at Columbia University published in Nature.

RNA-guided DNA insertion with CRISPR-associated transposases by Strecker et al., from the Feng Zhang's lab at the Broad Institute published in Science.

Recruitment of CRISPR-Cas systems by Tn7-like transposons by Peters et al., from the Eugene Koonin's lab at the NIH published in PNAS.

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January 6th, 2021

A novel combination of Class-I fumarases and metabolites (alpha-ketoglutarate and fumarate) signal the DNA damage response in E.coli

 

The functional link between metabolic genes, metabolites and DNA damage repair is largely unknown. Fumarases play roles both in the TCA cycle and the DNA damage response (DDR).

In this paper, Silas et al. from the Ophry Pines' lab at the Hebrew University and National University of Singapore study the DDR-associated functions of the three fumarase genes (named fumA, fumB and fumC) encoded in E.coli. The authors show that fumA and fumB deletion, but not fumC deletion, sensitize E.coli to the alkylating DNA damaging agent MMS and to ionizing radiation. Next, the authors use elegant complementation genetic experiments to demonstrate that E.coli fumarase A and B enzymes substitute yeast fumarase by restoring the DNA damage sensitivity of yeast cells deficient in fumarase to the DNA replication stress inducing agent hydroxyurea. It is unclear from the presented experiments whether yeast cells deficient for fum1 (fum1Δ) are also sensitive to MMS and whether E.coli genes complement a potential MMS sensitivity in yeast. The authors also show that the MMS sensitivity in bacteria is dependent on fumA and fumB enzymatic activities.

 

 

 

 

 

 

 

 

 

Interestingly, the authors convincingly show that the MMS sensitivity (but not IR sensitivity!) of E.coli mutant cells is suppressed by alpha-ketoglutarate, but not by other metabolites such as malate, fumarate or succinate (fumarase catalyzes the hydratation of fumarate to L-malate) (See Figure). Alpha-ketoglutarate enhances the DNA repair enzyme AlkB which repairs alkylated DNA (1me-A and 3me-C). To evaluate the effect of metabolites on AlkB activity, the authors use in vitro and in vivo assays to measure AlkB activity at MMS-induced DNA lesions. These experiments show that succinate and fumarate inhibit AlkB activity and MMS sensitivity suggesting that the accumulation of metabolites (succinate and fumarate) compete with alpha-ketoglutarate to inhibit AlkB-mediated DNA damage repair.

This interesting work reveals interesting interplay between DNA repair, metabolic activities and metabolites; and the conserved evolution of these mechanisms between prokaryotes and lower eukaryotes.

Extended reads:

Local generation of fumarate promotes DNA repair through inhibition of histone H3 demethylation by Jiang et al., from the Zhimin Lu's lab at MD Anderson published in Nature Cell Biology.

Bacterial fumarase and L-malic acid are evolutionary ancient components of the DNA damage response by Singer et al., from the Ophry Pines' lab from the Hebrew University and the National University of Singapore published in eLife.

2-Oxoglutarate-dependent dioxygenases in cancer by Losman et al., from the William Kaelin Jr's lab at Dana Farber Cancer Institute published in Nature Reviews Cancer

Nuclear metabolism and the regulation of the epigenome by Boon et al., from the Raul Mostoslavsky's lab at the Broad Institute published in Nature Metabolism.

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January 5th, 2021

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In this paper, Muller et al., from the Nicholas Ingolia's lab at UC Berkeley utilize CRISPR interference (CRISPRi) to disrupt the expression of a library of target genes in a gRNA-specific manner. In addition, the authors utilize transcriptional expression reporters that contain short-sequence barcodes associated with each CRISPRi gRNA. The combination of CRISPRi and barcoded expression reporters creates a new technology named CiBER-seq (See figure). This breakthrough technology enables a quantitative assessment of transcriptional changes on user-defined promoters, measured by the change in abundance of the barcodes, that is induced by the genome wide library of CRISPRi gRNAs. The authors used their technology to determine the transcriptional profiles of multiple promoters involved in diverse cellular processes, demonstrating the power of CiBER-seq to dissect regulatory pathways with transcriptional changes as readout.

 

Although this technology has only been tested in yeast, it is reasonable to believe that the implementation in mammalian systems will unlock major breakthroughs.

Extended reads:

Genome-wide, time-sensitive interrogation of the heat shock response under diverse stressors via ReporterSeq by Alford et al., from the Onn Brandman's lab at Stanford University published in BioRXiv.

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response by Adamson et al., from the Jonathan Weissman's lab at UCSF published in Cell.

Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens by Dixit et al., from the Aviv Regev's lab at the Broad Institute published in Cell.

Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq by Jaitin et al., from the Ido Amit's lab from the Weizmann Institute published in Cell.

Pooled CRISPR screening with single-cell transcriptome readout by Datlinger et al., from the Christoph Bock's lab at the Medical University of Vienna published in Nature Methods.

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January 4th, 2021

CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes

High-throughput genome wide CRISPR screens have revolutionized genetic experiments. Profiling systematic quantitative changes in gene expression across a population of cells with individual targeted genetic perturbations is challenging. The development of a method enabling the connection between a library of perturbations to their transcriptional phenotype would be extremely powerful to dissect genetic regulatory networks in massively parallel screens. 

January 3rd, 2021

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Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy

CRISPR-Cas13 are RNA-guided adaptive immune systems directed against single-stranded RNA. Upon RNA-guided detection of specific RNA molecules, Cas13 indiscriminately cleave all surrounding RNA molecules through its collateral trans-cleavage activity. This collateral activity has been harnessed has potent viral diagnostics (a.k.a. CRISPR diagnostics) by degrading quenched fluorescent RNA reporters (See figure).

In this paper, Fozouni et al. from the Daniel Fletcher's and Melanie Ott's labs at the Gladstone Institutes optimize CRISPR diagnostics for the detection of the SARS-CoV-2 RNA genome using Cas13. In this study, they show that a combination of multiple gRNAs targeting various segments of the SARS-CoV-2 genome increases the sensitivity of their detection assay. This is achieved by directly targeting pre-extracted RNA from nasal swabs of SARS-CoV-2 positive patients without pre-amplification of the viral material. Additionally, they show that the quantitative detection by Cas13 is proportional to the viral loads suggesting that this approach could potentially monitor infectiousness and disease progression. Finally, the authors derive a reader device based on a mobile phone camera as a portable plate reader. This could enable rapid, cost-effective and easy point-of-care detection ​for SARS-CoV-2.

Fast detection of pathogens outside of hospitals with limited need for laboratory equipments is critical to prevent the spread of highly infectious viruses among the population.

 

Extended reads:

Detection of SARS-CoV-2 with SHERLOCK One-Pot Testing by Joung et al., from the Feng Zhang's lab at the Broad Institute published in The New England Journal of Medicine.

Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2 from the Cameron Myhrvold's lab at the Broad Institute published in BioRXiv.

Nucleic acid detection with CRISPR-Cas13a/C2c2 from the Feng Zhang's lab at the Broad Institute published in Science.

January 2nd, 2021

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In vivo diversification of target genomic sites using processive base deaminase fusions blocked by dCas9

 

Continuous directed protein evolution enables diversification and selection of protein with improved functions.

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In this paper, Alvarez et al. from the Fernandez lab at the CNB-CSIC in Madrid develop the T7-targeted dCas9-limited in vivo mutagenesis (T7-DIVA) system (See figure). T7-DIVA corresponds to a fusion of deaminases (cytidine or adenine deaminases) with the T7 RNA polymerase. This fusion protein generates a mutagenic enzyme capable of inducing transition mutations within target genes. Interestingly, the authors placed the T7 promoter, that is recognized by T7-DIVA, at the 3'-end of the target gene in a reverse orientation, so that the promoter does not alter the expression of the target gene. The authors also elegantly used dCas9 arrays to block the elongation of the mutagenic enzyme in order to restrict the processivity of their mutagenic system in a gRNA-dependent manner.

This nice work extends the number of continuous directed molecular evolution systems for biotechnology and engineering applications.

Extended reads:

An orthogonal DNA replication system in yeast by Ravikumar et al.,  from the Chang Liu's lab at UC Irvine published in Nature Chemical Biology.

Methods for the directed evolution of proteins by Packer and Liu from the David Liu's lab at Harvard University published in Nature Reviews Genetics.

A Processive Protein Chimera Introduces Mutations across Defined DNA Regions In Vivo by Moore et al., from the Matthew Shoulders' lab at MIT published in the Journal of the American Chemical Society.

Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor by Chen et al., from the Fei Chen's lab at the Broad Insitute published in Nature Biotechnology.

Sequential Activation of Guide RNAs to Enable Successive CRISPR-Cas9 Activities

 

The ability to control the activation of Cas9 activity for genome editing by providing a series of instructions independently of cell states or exogenous inputs would transform multiplexed genome engineering.

In this paper, Clarke et al., from the Bradley Merrill's lab at the University of Illinois at Chicago succeeded in building such system. To this end, the authors introduce a self-cleaving ribozyme sequence within the gRNA hairpins in order to conserve the gRNA in an inactivate state (called proGuide for the analogy with prodrugs). Consequently, proGuides self-cleave resulting in gRNA inactivation. The authors' clever approach relies on secondary gRNAs (called activating-Guides or aGuides) which cleave at the ribozyme DNA sequence (either integrated within genomes or on episomal plasmids) to induce the deletion of the ribozyme sequence, thereby converting proGuides into activated gRNAs (called matureGuide). Interestingly, to stimulate a precise excision of the ribozyme the authors flanked the ribozyme sequence with sequences that favor repair by microhomology-mediated end joining in human cells. The authors also show how the conversion of a proGuide into a matureGuide can be used as a signal to convert other proGuides, thereby enabling sequential cascade of linear or ramified events.

This work establishes and provides elegant a proof-of-principle that an assembly of proGuides enables sequential control of Cas9 mobilization over time. 

Extended reads:

Continuous genetic recording with self-targeting CRISPR-Cas in human cells by Perli et al., from the Timothy Lu's lab at the MIT published in Science.

Rapidly evolving homing CRISPR barcodes by Kalhor et al., from the Prashant Mali and George Church labs from UC San Diego and MIT published in Nature Methods.

Emerging applications for DNA writers and molecular recorders from Farzadfard and Lu from the MIT published in Science.

January 1st, 2021