Wild-type Cas9 Research Articles

Abstract P23: Mitochondrial Delivery of Functional Nucleic Acids for Targeting of Mitochondrial Dysfunction

Abstract Mitochondria is the main power supply for cellular activity and a key player for the regulation of cellular metabolism, and they contain their own DNA. Critical proteins for mitochondrial functions are not only encoded by nuclear DNA (nDNA) but also by mitochondrial DNA (mtDNA). Abnormalities in mitochondrial functions could lead to diseases including neurodegeneration, diabetes, and cancer progression. The ability for a non-coding RNA (β2.7) of human cytomegalovirus (CMV) to localize to the host mitochondria revealed its potential to be used as a tool for mitochondria gene targeting. Using computational methods, our lab identified four thermodynamically conserved structural subdomains (D1 to D4) within the β2.7 RNA. While all four domains exhibited mitochondrial uptake potential, sub-domain D3 exhibited the highest absolute mitochondrial targeting activity and sub-domain D2 the highest activity per nucleotide. Further modifications on different sub-domain alignments found that maximum uptake efficiency was achieved by tandem repeats of 4 copies of D3 domains followed by 4 copies of D2 domains (abbreviated as D3 × 4_D2 × 4). Delivery of a GFP-coding mRNA or antisense RNA targeting mitochondrial genes mt-ATP6 and mt-ATP8 both confirmed its ability to co-deliver functional RNA into mitochondria. To cleave and destroy mutated mitochondrial genomes for future gene therapeutic applications, we explore mitochondrial delivery of the CRISPR-Cas9 system. The mRNAs coding for the wild-type Cas9 as well as more specific Cas9 variants were fused to the mitochondrial delivery vector (D3 × 4_D2 × 4). Latest RT-qPCR on mitochondrial RNA showed delivery of Cas9 mRNA into mitochondria of HepG2 cells. Currently, we investigate co-delivery of single-guide RNA and quantify Cas9 expression in mitochondria. In an alternative approach we deliver sequences coding for functional mitochondrial protein. Examples are the mRNAs coding for mitochondrial proteins mt-ND1, -ND4 or -ND6 which are frequently mutated is patients with Leber’s hereditary optic neuropathy (LHON). Citation Format: Jinglin Ruan, Volker Patzel. Mitochondrial Delivery of Functional Nucleic Acids for Targeting of Mitochondrial Dysfunction [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P23.

Cas9 is mostly orthogonal to human systems of DNA break sensing and repair.

CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo, Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair.

Lipid Nanoparticle-Mediated Gene Editing of Human Primary T Cells and Off-Target Analysis of the CRISPR-Cas9 Indels

Background and Aims: The CRISPR/Cas9 system has emerged as a powerful tool for gene editing of primary cells. In our previous work, we demonstrated a novel lipid nanoparticle (LNP) reagent for the multi-step engineering of gene-edited CAR T cells. We showed high cell viabilities and potent CAR-T mediated killing, even after multiple genetic manipulations. Here, we extend this work by assessing potential off-target editing effects in both LNP-treated T cells and T-cells where the CRISPR reagents were delivered by electroporation. Further, we evaluated multiple Cas9 variants and guide RNA targets. Methods: LNPs encapsulating wild type or high fidelity S.p. Cas9 mRNA and various TRAC and CD52 targeted guide RNAs (sgRNAs) were produced using our scalable NanoAssemblrTM microfluidics platform. Concurrently, electroporation was performed to deliver equivalent cargoes. Purified primary T cells were cultured, activated, and expanded in serum-free media in plates, flasks, or small bioreactors. The LNPs were added to cells by direct addition for gene editing. Gene expression and cell viability were measured using flow cytometry or colorimetric assays. Multi-target performance of CRISPR-Cas9 editing was evaluated through rhAmpSeq-based targeted next-generation sequencing (NGS) and indels analyzed for with CRISPRAltRations. Results: TCR or CD52 targeted Cas9 mRNA-LNP addition or electroporation yielded high single and double knockout efficiencies. High-throughput NGS analysis showed strong agreeance to flow cytometry for on-target analysis. We tested a range of sgRNA targets, wild-type and high-fidelity Cas9 mRNAs, and determined off-target editing for all targets and variants investigated. Similar results were obtained when comparing different LNP batch sizes (micro to milligram RNA) and cell culture vessels (0.1 to 45 million cells), demonstrating scalability of both LNP production and cell treatment. Conclusions: The results from this study further support the utility RNA-LNPs for the genetic engineering of primary T cells. The simple and gentle nature of LNP cell treatment allows for multiple genetic engineering steps for simultaneous expression and deletion of proteins for the next cell therapies. These LNPs can be easily manufactured from small-scale screening of RNA libraries to rapid scale-up for clinical translation.

Optimized whole-genome CRISPR interference screens identify ARID1A-dependent growth regulators in human induced pluripotent stem cells.

Perturbing expression is a powerful way to understand the role of individual genes, but can be challenging in important models. CRISPR-Cas screens in human induced pluripotent stem cells (iPSCs) are of limited efficiency due to DNA break-induced stress, while the less stressful silencing with an inactive Cas9 has been considered less effective so far. Here, we developed the dCas9-KRAB-MeCP2 fusion protein for screening in iPSCs from multiple donors. We found silencing in a 200bp window around the transcription start site in polyclonal pools to be as effective as using wild-type Cas9 for identifying essential genes, but with much reduced cell numbers. Whole-genome screens to identify ARID1A-dependent dosage sensitivity revealed the PSMB2 gene, and enrichment of proteasome genes among the hits. This selective dependency was replicated with a proteasome inhibitor, indicating a targetable drug-gene interaction. Many more plausible targets in challenging cell models can be efficiently identified with our approach.

Prime editing with genuine Cas9 nickases minimizes unwanted indels

Unlike CRISPR-Cas9 nucleases, which yield DNA double-strand breaks (DSBs), Cas9 nickases (nCas9s), which are created by replacing key catalytic amino-acid residues in one of the two nuclease domains of S. pyogenesis Cas9 (SpCas9), produce nicks or single-strand breaks. Two SpCas9 variants, namely, nCas9 (D10A) and nCas9 (H840A), which cleave target (guide RNA-pairing) and non-target DNA strands, respectively, are widely used for various purposes, including paired nicking, homology-directed repair, base editing, and prime editing. In an effort to define the off-target nicks caused by these nickases, we perform Digenome-seq, a method based on whole genome sequencing of genomic DNA treated with a nuclease or nickase of interest, and find that nCas9 (H840A) but not nCas9 (D10A) can cleave both strands, producing unwanted DSBs, albeit less efficiently than wild-type Cas9. To inactivate the HNH nuclease domain further, we incorporate additional mutations into nCas9 (H840A). Double-mutant nCas9 (H840A + N863A) does not exhibit the DSB-inducing behavior in vitro and, either alone or in fusion with the M-MLV reverse transcriptase (prime editor, PE2 or PE3), induces a lower frequency of unwanted indels, compared to nCas9 (H840A), caused by error-prone repair of DSBs. When incorporated into prime editor and used with engineered pegRNAs (ePE3), we find that the nCas9 variant (H840A + N854A) dramatically increases the frequency of correct edits, but not unwanted indels, yielding the highest purity of editing outcomes compared to nCas9 (H840A).

Improving the on-target activity of high-fidelity Cas9 editors by combining rational design and random mutagenesis.

Genomic and post-genomic editors based on CRISPR/Cas systems are widely used in basic research and applied sciences, including human gene therapy. Most genome editing tools are based on the CRISPR/Cas9 type IIA system from Streptococcus pyogenes. Unfortunately, a number of drawbacks have hindered its application in therapeutic approaches, the most serious of which is the relatively high level of off-targets. To overcome this obstacle, various high-fidelity Cas9 variants have been created. However, they show reduced on-target activity compared to wild-type Cas9 possibly due to increased sensitivity to eukaryotic chromatin. Here, we combined a rational approach with random mutagenesis to create a set of new Cas9 variants showing high specificity and increased activity in Saccharomyces cerevisiae yeast. Moreover, a novel mutation in the PAM (protospacer adjacent motif)-interacting Cas9 domain was found, which increases the on-target activity of high-fidelity Cas9 variants while retaining their high specificity. The obtained data suggest that this mutation acts by weakening the eukaryotic chromatin barrier for Cas9 and rearranging the RuvC active center. Improved Cas9 variants should further advance genome and post-genome editing technologies. KEY POINTS: • D147Y and P411T mutations increase the activity of high-fidelity Cas9 variants. • The new L1206P mutation further increases the activity of high-fidelity Cas9 variants. • The L1206P mutation weakens the chromatin barrier for Cas9 editors.

Comparison of DNA targeting CRISPR editors in human cells

BackgroundProfiling and comparing the performance of current widely used DNA targeting CRISPR systems provide the basic information for the gene-editing toolkit and can be a useful resource for this field. In the current study, we made a parallel comparison between the recently reported miniature Cas12f1 (Un1Cas12f1 and AsCas12f1) and the widely used Cas12a and Cas9 nucleases in mammalian cells.ResultsWe found that as a CRISPRa activator, Un1Cas12f1 could induce gene expression with a comparable level to that of Cas12a and Cas9, while as a DNA cleavage editor, Cas12f1 exhibited similar properties to Cas12a, like high specificity and dominantly induced deletions over insertions, but with less activity. In contrast, wild-type SpCas9 showed the highest activity, lowest specificity, and induced balanced deletions and insertions. Thus, Cas12f1 is recommended for gene-activation-based applications, Cas12a is for therapy applications, and wild-type Cas9 is for in vitro and animal investigations.ConclusionThe comparison provided the editing properties of the widely used DNA-targeting CRISPR systems in the gene-editing field.

A Test System for Assessment of the Activity of Mutant Cas9 Variants in Saccharomyces cerevisiae

The key component of the revolutionary Streptococcus pyogenes CRISPR/Cas genome editing technology is the multidomain protein Cas9. However, the specificity of wild type Cas9 is not sufficiently high for editing large genomes of higher eukaryotes, which limits the realization of the potential of genomic editing both in fundamental investigations and in the therapy of genetic diseases. The main way to obtain more specific variants of Cas9 is through mutagenesis followed by characterization of mutant proteins in in vitro or in vivo test systems. The in vitro and some in vivo test systems described in the literature are often labor-intensive and have scaling limitations, which makes it challenging to screen SpCas9 mutant variant libraries. In order to develop a simple method for high-throughput screening of Cas9 mutants in vivo, we characterized three test systems using CRISPR/Cas9-mediated inactivation of the reporter genes, tsPurple, ADE2, and URA3, in the Saccharomyces cerevisiae yeast as a model subject. We measured the activities of high-precision forms of Cas9, evoCas9, and HiFiCas9, and compared them with the wild-type form. ADE2 gene inactivation was found to be the most valid method for the evaluation of Cas9 activity. In the test-system developed, the sensitivity to chromatin structure was demonstrated for the high-fidelity variant of Cas9, HiFiCas9. The proposed test-system can be used for the development of new generation genome editors.

Cas9-mediated endogenous plasmid loss in Borrelia burgdorferi.

The spirochete Borrelia burgdorferi, which causes Lyme disease, has the most segmented genome among known bacteria. In addition to a linear chromosome, the B. burgdorferi genome contains over 20 linear and circular endogenous plasmids. While many of these plasmids are dispensable under in vitro culture conditions, they are maintained during the natural life cycle of the pathogen. Plasmid-encoded functions are required for colonization of the tick vector, transmission to the vertebrate host, and evasion of host immune defenses. Different Borrelia strains can vary substantially in the type of plasmids they carry. The gene composition within the same type of plasmid can also differ from strain to strain, impeding the inference of plasmid function from one strain to another. To facilitate the investigation of the role of specific B. burgdorferi plasmids, we developed a Cas9-based approach that targets a plasmid for removal. As a proof-of-principle, we showed that targeting wild-type Cas9 to several loci on the endogenous plasmids lp25 or lp28-1 of the B. burgdorferi type strain B31 results in sgRNA-specific plasmid loss even when homologous sequences (i.e., potential sequence donors for DNA recombination) are present nearby. Cas9 nickase versions, Cas9D10A or Cas9H840A, also cause plasmid loss, though not as robustly. Thus, sgRNA-directed Cas9 DNA cleavage provides a highly efficient way to eliminate B. burgdorferi endogenous plasmids that are non-essential in axenic culture.

Quantitative analysis of CRISPR/Cas9-mediated provirus deletion in blue egg layer chicken PGCs by digital PCR

Primordial germ cells (PGCs), the precursors of sperm and oocytes, pass on the genetic material to the next generation. The previously established culture system of chicken PGCs holds many possibilities for functional genomics studies and the rapid introduction of desired traits. Here, we established a CRISPR/Cas9-mediated genome editing protocol for the genetic modification of PGCs derived from chickens with blue eggshell color. The sequence targeted in the present report is a provirus (EAV-HP) insertion in the 5’-flanking region of the SLCO1B3 gene on chromosome 1 in Araucana chickens, which is supposedly responsible for the blue eggshell color. We designed pairs of guide RNAs (gRNAs) targeting the entire 4.2 kb provirus region. Following transfection of PGCs with the gRNA, genomic DNA was isolated and analyzed by mismatch cleavage assay (T7EI). For absolute quantification of the targeting efficiencies in homozygous blue-allele bearing PGCs a digital PCR was established, which revealed deletion efficiencies of 29% when the wildtype Cas9 was used, and 69% when a high-fidelity Cas9 variant was employed. Subsequent single cell dilutions of edited PGCs yielded 14 cell clones with homozygous deletion of the provirus. A digital PCR assay proved the complete absence of this provirus in cell clones. Thus, we demonstrated the high efficiency of the CRISPR/Cas9 system in introducing a large provirus deletion in chicken PGCs. Our presented workflow is a cost-effective and rapid solution for screening the editing success in transfected PGCs.

WT-PE: Prime editing with nuclease wild-type Cas9 enables versatile large-scale genome editing

Large scale genomic aberrations including duplication, deletion, translocation, and other structural changes are the cause of a subtype of hereditary genetic disorders and contribute to onset or progress of cancer. The current prime editor, PE2, consisting of Cas9-nickase and reverse transcriptase enables efficient editing of genomic deletion and insertion, however, at small scale. Here, we designed a novel prime editor by fusing reverse transcriptase (RT) to nuclease wild-type Cas9 (WT-PE) to edit large genomic fragment. WT-PE system simultaneously introduced a double strand break (DSB) and a single 3′ extended flap in the target site. Coupled with paired prime editing guide RNAs (pegRNAs) that have complementary sequences in their 3′ terminus while target different genomic regions, WT-PE produced bi-directional prime editing, which enabled efficient and versatile large-scale genome editing, including large fragment deletion up to 16.8 megabase (Mb) pairs and chromosomal translocation. Therefore, our WT-PE system has great potential to model or treat diseases related to large-fragment aberrations.

Rational Engineering of CRISPR-Cas9 Nuclease to Attenuate Position-Dependent Off-Target Effects.

The RNA-guided Cas9 nuclease from Streptococcus pyogenes has become an important gene-editing tool. However, its intrinsic off-target activity is a major challenge for biomedical applications. Distinct from some reported engineering strategies that specifically target a single domain, we rationally introduced multiple amino acid substitutions across multiple domains in the enzyme to create potential high-fidelity variants, considering the Cas9 specificity is synergistically determined by various domains. We also exploited our previously derived atomic model of activated Cas9 complex structure for guiding new modifications. This approach has led to the identification of the HSC1.2 Cas9 variant with enhanced specificity for DNA cleavage. While the enhanced specificity associated with the HSC1.2 variant appeared to be position-dependent in the in vitro cleavage assays, the frequency of off-target DNA editing with this Cas9 variant is much less than that of the wild-type Cas9 in human cells. The potential mechanisms causing the observed position-dependent effect were investigated through molecular dynamics simulation. Our discoveries establish a solid foundation for leveraging structural and dynamic information to develop Cas9-like enzymes with high specificity in gene editing.

Key sequence features of CRISPR RNA for dual-guide CRISPR-Cas9 ribonucleoprotein complexes assembled with wild-type or HiFi Cas9.

Specific sequence features of the protospacer and protospacer-adjacent motif (PAM) are critical for efficient cleavage by CRISPR-Cas9, but current knowledge is largely derived from single-guide RNA (sgRNA) systems assessed in cultured cells. In this study, we sought to determine gRNA sequence features of a more native CRISPR-Cas9 ribonucleoprotein (RNP) complex with dual-guide RNAs (dgRNAs) composed of crRNA and tracrRNA, which has been used increasingly in recent CRISPR-Cas9 applications, particularly in zebrafish. Using both wild-type and HiFi SpCas9, we determined on-target cleavage efficiencies of 51 crRNAs in zebrafish embryos by assessing indel occurrence. Statistical analysis of these data identified novel position-specific mononucleotide features relevant to cleavage efficiencies throughout the protospacer sequence that may be unique to CRISPR-Cas9 RNPs pre-assembled with perfectly matched gRNAs. Overall features for wild-type Cas9 resembled those for HiFi Cas9, but specific differences were also observed. Mutational analysis of mononucleotide features confirmed their relevance to cleavage efficiencies. Moreover, the mononucleotide feature-based score, CRISPR-kp, correlated well with efficiencies of gRNAs reported in previous zebrafish RNP injection experiments, as well as independently tested crRNAs only in RNP format, but not with Cas9 mRNA co-injection. These findings will facilitate design of gRNA/crRNAs in genome editing applications, especially when using pre-assembled RNPs.

An assessment of genome-editing efficiency of a newly developed Cas9 in C. elegans.

Minimizing the off-target effects of the programmable genome editing tool CRISPR/Cas9 is critical for its potential therapeutic applications. A recent study reported a new Cas9 variant, SuperFi-Cas9, that dramatically reduces off-target DNA cleavage while retaining on-target DNA cleavage activity in in vitro assays. Here we evaluated the genome editing potential of SuperFi-Cas9 by examining its efficiency in mutagenizing targeted genes in the nematode C. elegans . We found that gene mutagenesis rates induced by SuperFi-Cas9 through either non-homologous end joining or homology-directed repair were much lower than those by the wild type Cas9, indicating that Super-Cas9 had very low in vivo DNA cleavage activity. Our results also suggest that C. elegans may serve as an excellent model system for assessing in vivo genome-editing efficiency of newly-developed Cas9 variants.

Probing the stability of the SpCas9-DNA complex after cleavage.

CRISPR–Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 and its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different Streptococcus pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After initial target strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability than the pre-cleavage state. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around Cas9 bound to the target strand. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress.

Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis.

There have been many engineered Cas9 variants that were developed to minimize unintended cleavage of off-target DNAs, but detailed mechanism for the way they regulate the target specificity through DNA:RNA heteroduplexation remains poorly understood. We used single-molecule FRET assay to follow the dynamics of DNA:RNA heteroduplexation for various engineered Cas9 variants with respect to on-target and off-target DNAs. Just like wild-type Cas9, these engineered Cas9 variants exhibit a strong correlation between their conformational structure and nuclease activity. Compared with wild-type Cas9, the fraction of the cleavage-competent state dropped more rapidly with increasing base-pair mismatch, which gives rise to their enhanced target specificity. We proposed a reaction model to quantitatively analyze the degree of off-target discrimination during the successive process of R-loop expansion. We found that the critical specificity enhancement step is activated during DNA:RNA heteroduplexation for evoCas9 and HypaCas9, while it occurs in the post-heteroduplexation stage for Cas9-HF1, eCas9, and Sniper-Cas9. This study sheds new light on the conformational dynamics behind the target specificity of Cas9, which will help strengthen its rational designing principles in the future.

Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations.

Microsatellite expansions are the cause of >20 neurological or developmental human disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned as a gene editing approach. Here, we measured the efficacy of several CRISPR–Cas nucleases to induce recombination within disease-related microsatellites, in Saccharomyces cerevisiae. Broad variations in nuclease performances were detected on all repeat tracts. Wild-type Streptococcus pyogenes Cas9 (SpCas9) was more efficient than Staphylococcus aureus Cas9 on all repeats tested, except (CAG)33. Cas12a (Cpf1) was the most efficient on GAA trinucleotide repeats, whereas GC-rich repeats were more efficiently cut by SpCas9. The main genetic factor underlying Cas efficacy was the propensity of the recognition part of the sgRNA to form a stable secondary structure, independently of its structural part. This suggests that such structures form in vivo and interfere with sgRNA metabolism. The yeast genome contains 221 natural CAG/CTG and GAA/CTT trinucleotide repeats. Deep sequencing after nuclease induction identified three of them as carrying statistically significant low frequency mutations, corresponding to SpCas9 off-target double-strand breaks.

Controlling Hematopoietic Stem Cell Quiescence By Ablating the TGFβ-Responsive, Hematopoietic-Hpecific Enhancer of CDKN1C (p57)

Introduction: The cyclin-dependent kinase inhibitor (CDKI) CDKN1C (p57) is a putative tumor suppressor gene with strong differential expression in both human and murine hematopoietic stem cells (HSCs). The most immature, quiescent HSCs are marked by p57 expression and p57 is rapidly downregulated as HSCs enter cell cycle and differentiate. Genetic deletion of p57 leads to abnormal HSC cell cycle entry and, in murine models, a propensity for HSC exhaustion indicating that p57 is an essential gatekeeper of HSC function. TGFβ signaling is an important mediator of HSC quiescence and mandates the withdrawal of HSCs from cell cycle following hematopoietic stress. We have found that p57 is a direct transcriptional target of TGFβ in HSCs where it serves as an essential downstream mediator of TGFβ signaling. In HSCs, genetic deletion of p57 phenocopies the effects of TGFβ blockade in vivo. The regulation of p57 is complex and interesting. In almost all epithelial cell types, p57 is poorly expressed and is not a transcriptional target of TGFβ. This is also true for most differentiated hematopoietic lineages. The mechanism for the tissue-type and differentiation stage-specific expression of p57 and its control by TGFβ in HSCs is not known. We hypothesized that a hematopoietic enhancer for p57 must be responsible for TGFβ induction in HSCs.Methods: We performed chromatin immunoprecipitation (ChIP/ChIP-chip/ChIP-seq) in human, AML-derived M091 cells that faithfully upregulate p57 in response to TGFβ signaling in a manner indistinguishable from human cord blood derived CD34+ cells. Cultured cells were studied while proliferating and following treatment with TGFβ for various times. ChIP was performed for histone modifications that mark enhancer elements (histone H3 lysine 4 mono-methylation, H3K4me1, and H3K27-acetlylation, H3K27ac) and promoters (histone H3 lysine 4 tri-methylation, H3K4me2, and H3K27-acetlylation, H3K27ac). Genomic localization of receptor activated phospho-Smad2 was also identified before and after TGFβ stimulation. Putative enhancers were cloned into reporter constructs that were then studied after stable incorporation into the genome of M091 and K562 cells.Results: We identified putative enhancer elements more than 100 KB from the p57 gene body (Fig. A). A cluster of three small elements fulfilled our biochemical criterion for a hematopoietic-specific enhancer: enhancer histone marks (H3K4me1/H3K27ac); hyper-acetylation and phospho-Smad2 binding in response to TGFβ (Fig. B, D). When subcloned and tested in reporter assays, the elements had enhancer function and two of the three were induced by TGFβ (Fig. C). Interestingly, the ENCODE consortium identified the genomic region of our putative enhancers as overlapping with a DNAseI hypersensitive site and harboring the chromatin signature of a “Strong Enhancer” only in hematopoietic cells (K562) but not in other cell type tested. Nonetheless, unequivocal classification of this element as a p57 enhancer required in vivo analysis of HSCs with targeted deletion of the enhancer element.We designed and cloned, ten guide-RNAs (gRNAs) targeting regions upstream and downstream of this putative enhancer cluster. These gRNAs were then tested in HeLa cells by transient transfection with wild-type Cas9 nuclease. Using this approach, we selected a pair of gRNAs with the highest efficiency in deleting the entire enhancer cluster as assessed by ddPCR (~30% alleles). These gRNAs or non-targeting control gRNAs and Cas9 were then transfected into M091 cells along with a GFP reporter. Transfected GFP+ cells were then sorted 36 hrs later. A second lot of unsorted cells were grown in bulk cultures. Allelic ratios of this enhancer element were tracked by ddPCR to assess the relative outgrowth of genomically unmodified cells compared to those that had this putative p57 enhancer deleted. The targeted MO91 population has exhibited resistance to TGFβ as assessed using a growth curve assay.Conclusions: A hematopoietic-specific enhancer lying ~100 kb from the p57 gene body is important for TGFβ-mediated upregulation of p57 and consequent cell cycle withdrawal in immature hematopoietic cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cas9 deactivation with photocleavable guide RNAs

Precise control of CRISPR-Cas9 would improve its safety and applicability. Controlled CRISPR inhibition is a promising approach but is complicated by separate inhibitor delivery, incomplete deactivation, and slow kinetics. To overcome these obstacles, we engineered photocleavable guide RNAs (pcRNAs) that endow Cas9 nucleases and base editors with a built-in mechanism for light-based deactivation. pcRNA enabled the fastest (<1min) and most complete (<1% residual indels) approach for Cas9 deactivation. It also exhibited significantly enhanced specificity with wild-type Cas9. Time-resolved deactivation revealed that 12-36h of Cas9 activity or 2-4h of base editor activity was sufficient to achieve high editing efficiency. pcRNA is useful for studies of the cellular response to DNA damage by abolishing sustained cycles of damage and repair that would otherwise desynchronize response trajectories. Together, pcRNA expands the CRISPR toolbox for precision genome editing and studies of DNA damage and repair.

A “Shot in the Arm” for Sickle Cell Disease

A “Shot in the Arm” for Sickle Cell Disease