BackgroundSingle-guide RNA (sgRNA) is one of the two key components of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system. The current commonly used sgRNA structure has a shortened duplex compared with the native bacterial CRISPR RNA (crRNA)–transactivating crRNA (tracrRNA) duplex and contains a continuous sequence of thymines, which is the pause signal for RNA polymerase III and thus could potentially reduce transcription efficiency.ResultsHere, we systematically investigate the effect of these two elements on knockout efficiency and showed that modifying the sgRNA structure by extending the duplex length and mutating the fourth thymine of the continuous sequence of thymines to cytosine or guanine significantly, and sometimes dramatically, improves knockout efficiency in cells. In addition, the optimized sgRNA structure also significantly increases the efficiency of more challenging genome-editing procedures, such as gene deletion, which is important for inducing a loss of function in non-coding genes.ConclusionsBy a systematic investigation of sgRNA structure we find that extending the duplex by approximately 5 bp combined with mutating the continuous sequence of thymines at position 4 to cytosine or guanine significantly increases gene knockout efficiency in CRISPR-Cas9-based genome editing experiments.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0846-3) contains supplementary material, which is available to authorized users.

Endoplasmic reticulum (ER)-associated degradation (ERAD) represents a principle quality control mechanism to clear misfolded proteins in the ER; however its physiological significance and the nature of endogenous ERAD substrates remain largely unexplored. Here we discover that IRE1α, the sensor of unfolded protein response (UPR), is a bona fide substrate of the Sel1L-Hrd1 ERAD complex. ERAD-mediated IRE1α degradation occurs under basal conditions in a BiP-dependent manner, requires both intramembrane hydrophilic residues of IRE1α and lectin protein OS9, and is attenuated by ER stress. ERAD deficiency causes IRE1α protein stabilization, accumulation and mild activation both in vitro and in vivo. Although enterocyte-specific Sel1L-knockout mice (Sel1LΔIEC) are viable and appear normal, they are highly susceptible to experimental colitis and inflammation-associated dysbiosis, in an IRE1α-dependent but CHOP-independent manner. Hence, Sel1L-Hrd1 ERAD serves a distinct, essential function in restraint of IRE1α signaling in vivo by managing its protein turnover.

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Summary West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the endoplasmic reticulum-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death.

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Microprocessor [Drosha-DGCR8 (DiGeorge syndrome critical region gene 8) complex] processing of primary microRNA (pri-miRNA) is the critical first step in miRNA biogenesis, but how the Drosha cleavage site is determined has been unclear. Previous models proposed that the Drosha-DGCR8 complex measures either ∼22 nt from the upper stem-single-stranded RNA (ssRNA, terminal loop) junction or ∼11 nt from the lower stem-ssRNA junction to determine the cleavage site. Here, using miRNA-offset RNAs to determine the Drosha cleavage site, we show that the Microprocessor measures the distances from both the lower and upper stem-ssRNA junctions to determine the cleavage site in human cells, and optimal distances from both structures are critical to the precision of Drosha processing. If the distances are not optimal, Drosha tends to cleave at multiple sites, which can, in turn, generate multiple 5′ isomiRs. Thus, our results also reveal a mechanism of 5′ isomiR generation.moR | DcRNA | alternative Drosha processing I n mammals, the canonical pathway of miRNA biogenesis is initiated by the Drosha-DGCR8 (DiGeorge syndrome critical region gene 8) complex (the Microprocessor), which processes long primary miRNAs (pri-miRNAs) into ∼60-nt pre-miRNAs for further processing by Dicer into a duplex ∼22 nt long. One or both strands of the duplex are loaded into the RNA-induced silencing complex (RISC) to repress target gene expression (1, 2). Although the major players of miRNA biogenesis are mostly known, no model precisely predicts how the miRNA biogenesis machinery recognizes and processes a novel pri-miRNA or an miRNA-mimicking shRNA and which strand will finally be loaded into RISC. The missing details in miRNA biogenesis are major obstacles to rational design of miRNA-based shRNA or artificial miRNA (amiRNA) that generates predictable results, as this kind of amiRNA must be processed by the endogenous miRNA machinery to be functional. The lack of a precise model of miRNA biogenesis also makes it difficult to accurately predict novel miRNA genes in the genome. In fact, a recent study found that more than 150 miRNAs annotated in miRBase (of 564 miRNAs checked) were not miRNAs at all (3). Thus, there is still a need to improve our understanding of how the miRNA biogenesis machinery recognizes and processes pri-miRNA into mature miRNA.The secondary structure of a canonical pri-miRNA usually consists of four parts: a terminal loop, an upper stem that encompasses the mature miRNA duplex of approximately two helical turns, the lower stem, which is an approximate one-helical-turn extension of the miRNA duplex, and the basal segments, which are single-stranded flanking sequences (4, 5). In 2005, Zeng et al. proposed that a large terminal loop is required to guide Drosha cleavage to occur at ∼22 nt from the junction of the terminal loop single-stranded RNA (ssRNA) and upper stem (5). In addition, they showed that single-stranded flanking sequences around the miRNA hairpin structure are also required for efficient Drosha processing (6). Howe...