Precise transcript targeting by CRISPR-Csm complexes

Robust and precise transcript targeting in mammalian cells remains a difficult challenge using existing approaches due to inefficiency, imprecision and subcellular compartmentalization. Here we show that the clustered regularly interspaced short palindromic repeats (CRISPR)-Csm complex, a multiprotein effector from type III CRISPR immune systems in prokaryotes, provides surgical RNA ablation of both nuclear and cytoplasmic transcripts. As part of the most widely occurring CRISPR adaptive immune pathway, CRISPR-Csm uses a programmable RNA-guided mechanism to find and degrade target RNA molecules without inducing indiscriminate trans-cleavage of cellular RNAs, giving it an important advantage over the CRISPR-Cas13 family of enzymes. Using single-vector delivery of the Streptococcus thermophilus Csm complex, we observe high-efficiency RNA knockdown (90-99%) and minimal off-target effects in human cells, outperforming existing technologies including short hairpin RNA- and Cas13-mediated knockdown. We also find that catalytically inactivated Csm achieves specific and durable RNA binding, a property we harness for live-cell RNA imaging. These results establish the feasibility and efficacy of multiprotein CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes

Human eIF4AIII interacts with an eIF4G-like partner, NOM1, revealing an evolutionarily conserved function outside the exon junction complex

Despite the lack of an exon junction complex (EJC), Saccharomyces cerevisiae contains Fal1p, a DEAD-box helicase highly homologous to eIF4AIII. We show that yeast Fal1p is functionally orthologous to human eIF4AIII, since expression of human eIF4AIII complements both the lethal phenotype and the 18S rRNA biogenesis defect of fal1Δ(null) yeast. We further show that yeast Fal1p interacts genetically with an eIF4G-like protein, Sgd1p: One allele of sgd1 acts as a dominant extragenic suppressor of a mutation in a predicted RNA-binding residue of Fal1p, whereas another synthetically exacerbates the growth defect of this fal1 mutation. Both sgd1 mutations map to a single, short, evolutionarily conserved patch that matches key eIF4A-interacting residues of eIF4G when superimposed on the X-ray structure of the eIF4A/eIF4G complex. We demonstrate direct physical interactions between yeast Sgd1p and Fal1p, and between their human orthologs (NOM1 and eIF4AIII) in vitro and in vivo, identifying human NOM1 as a missing eIF4G-like interacting partner of eIF4AIII. Knockdown of eIF4AIII and NOM1 in human cells demonstrates that this novel conserved eIF4A/eIF4G-like complex acts in pre-rRNA processing, adding to the established functions of eIF4A/eIF4G in translation initiation and of eIF4AIII as the core component of the EJC

Decorating chromatin for enhanced genome editing using CRISPR-Cas9

CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile method to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K36me3 and H3K4me3 to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in the genome to increase the observed HDR efficiency by threefold and HDR:indel ratio by fivefold compared with that induced by unmodified Cas9. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin features for the balance between DNA repair mechanisms during CRISPR-Cas genome editing and provide a strategy to increase HDR efficiency

Genome-wide identification of autosomal genes with allelic imbalance of chromatin state

In mammals, monoallelic gene expression can result from X-chromosome inactivation, genomic imprinting, and random monoallelic expression (RMAE). Epigenetic regulation of RMAE is not fully understood. Here we analyze allelic imbalance in chromatin state of autosomal genes using ChIP-seq in a clonal cell line. We identify approximately 3.7% of autosomal genes that show significant differences between chromatin states of two alleles. Allelic regulation is represented among several functional gene categories including histones, chromatin modifiers, and multiple early developmental regulators. Most cases of allelic skew are produced by quantitative differences between two allelic chromatic states that belong to the same gross type (active, silent, or bivalent). Combinations of allelic states of different types are possible but less frequent. When different chromatin marks are skewed on the same gene, their skew is coordinated as a result of quantitative relationships between these marks on each individual allele. Finally, combination of allele-specific densities of chromatin marks is a quantitative predictor of allelic skew in gene expression

Relationships between the magnitudes of allelic skew of different marks on the same gene.

<p>For all genes with sufficient number of allelically assigned reads, allelic skews (log2 of <i>mus</i>:<i>cas</i> ratio) are plotted for one mark vs. another: <b>A,</b> K27me3 vs. K4me3; <b>B,</b> K36me3 vs. K4me3; <b>C,</b> POL2S2 vs. K4me3. Horizontal and vertical dashed lines mark the 2-fold allelic difference. The majority of genes do not show significant skew in any mark (gray points around origin). Most of the genes with skewed chromatin state (colored points) have a skew in only one mark and no significant skew in the other (purple triangles and magenta squares). However, when both marks are skewed (cyan circles), these skews are anticorrelated for active vs. repressive mark (A) and correlated for two active marks (B, C).</p

Quantitative relationships between active (K4me3) and repressive (K27me3) chromatin marks on the same promoter.

<p>Input-normalized densities of K4me3 and K27me3 in TSS-proximal regions of individual autosomal genes are shown in a two-dimensional scatter plot in log-log space, with color (black to red) representing levels of gene expression (silent to strongly active) measured by FPKM values based on RNA-Seq. Three major regions of the plot correspond to distinct levels and correlation patterns of K4me3 and K27me3. These regions largely correspond to silent (region 1), bivalent (region 2) and active genes (region 3).</p

Relationships between chromatin marks on individual alleles are similar to the relationships on the composite level.

<p><b>A, B</b>, Scatter plots of composite densities of K4me3 in TSS-proximal regions vs. (A) K27me3 in TSS-proximal regions and (B) K36me3 on gene bodies. Color represents composite levels of gene expression measured by FPKM values based on RNA-Seq. Active genes (upper part of the plots) show higher levels of active marks K4me3 and K36me3, general anticorrelation between K4me3 and K27me3, and positive correlation between K4me3 and K36me3. Permanently silenced genes (lower part of the plots) show depletion of K4me3 and K36me3, and the corresponding depletion of K27me3. Bivalent region is the area of highest K27me3 densities and intermediate K4me3 densities that connects active and permanently silenced branches in (A). <b>C, D,</b> Scatter plots of inferred mark densities on individual allele (maternal “M” allele as an example) for K4me3 in TSS-proximal regions vs. (C) K27me3 in TSS-proximal regions and (D) K36me3 on gene bodies. Color represents allelic levels of expression measured by FPKM values based on RNA-Seq.</p

Pairwise combinations of allelic chromatin states that result in observed allelic skew of a chromatin mark.

<p>Allelic promoters are shown as points in the space of K4me3 and K27me3 densities. <b>A,</b> Schematic of major types of allelic chromatin states and their possible combinations on two alleles. Regions 1, 2, and 3 correspond to silent, bivalent, and active alleles, respectively. Combinations observed in larger numbers of genes (“observed”) are shown as cyan lines. Combination observed in fewer genes (“unobserved”) is shown as orange line. <b>B,</b> Scatter plot of K4me3 vs. K27me3 promoter densities on maternal (M) <i>mus</i> allele of all autosomal genes shown as gray points; genes with allelic skew in K4me3 are highlighted in blue. Regions 1, 2, and 3 are marked by dashed lines. <b>C-E,</b> Scatter plots of K4me3 vs. K27me3 promoter densities on paternal (P) <i>cas</i> allele, shown as gray points. Red points in these three plots highlight three subgroups of genes shown in <b>B</b>: genes whose maternal allele belongs to silent, bivalent, or active type (regions 1, 2, and 3 in <b>B</b>, marked for the reference by a blue rectangle in each corresponding plot <b>C-E</b>). Hue indicates the local density of paternal alleles with similar K4me3/K27me3 densities. <b>C,</b> Among genes with K4me3 skew whose maternal allele is in a silent state (region 1 in <b>B,</b> <i>n</i> = 24), paternal allele (P) generally has medium to high K4me3 density and low K27me3 density. <b>D,</b> Among genes with K4me3 skew whose maternal allele is in a bivalent state (region 2 in <b>B,</b> <i>n</i> = 66), paternal allele (P) generally also resides in a bivalent state. <b>E,</b> Among genes with K4me3 skew whose maternal allele is in an active state (region 3 in <b>B,</b> <i>n</i> = 281), paternal allele (P) most often resides in an active or bivalent state; there are much fewer cases of a fully silent paternal allele.</p