Cpf1 nucleases were recently reported to be highly specific and programmable nucleases with efficiencies comparable to those of SpCas9. AsCpf1 and LbCpf1 require a single crRNA and recognize a 5′-TTTN-3′ protospacer adjacent motif (PAM) at the 5′ end of the protospacer for genome editing. For widespread application in precision site-specific human genome editing, the range of sequences that AsCpf1 and LbCpf1 can recognize is limited due to the size of this PAM. To address this limitation, we sought to identify a novel Cpf1 nuclease with simpler PAM requirements. Specifically, here we sought to test and engineer FnCpf1, one reported Cpf1 nuclease (FnCpf1) only requires 5′-TTN-3′ as a PAM but does not exhibit detectable levels of nuclease-induced indels at certain locus in human cells. Surprisingly, we found that FnCpf1 possesses DNA cleavage activity in human cells at multiple loci. We also comprehensively and quantitatively examined various FnCpf1 parameters in human cells, including spacer sequence, direct repeat sequence and the PAM sequence. Our study identifies FnCpf1 as a new member of the Cpf1 family for human genome editing with distinctive characteristics, which shows promise as a genome editing tool with the potential for both research and therapeutic applications.

BackgroundType 2 diabetes (T2D) is closely associated with liver fibrosis, but no effective treatments are currently available. This study was designed to investigate the therapeutic effects of ADSCs on insulin resistance, hyperglycemia, and liver fibrosis on T2D rats.MethodsWe first established a T2D rat model with liver fibrosis by using the combination of a high-fat diet (HFD), low-dose streptozotocin (STZ), and carbon tetrachloride (CCl4). Subsequently, the model rats were administrated by tail vein injection of PBS or ADSCs, respectively. Thereafter, insulin resistance and liver function were assessed by biochemical analysis, ELISA, histopathological examination, and q-PCR assay, respectively. Moreover, the molecular mechanisms of ADSCs on the effect of the TGF-β1/SMAD3 signaling pathway were further analyzed.ResultsOur data showed that ADSC transplantation significantly alleviated insulin resistance and hyperglycemia in the liver-injured T2D rats. We also found that ADSC transplantation could attenuate liver injury by improving liver function and inhibiting pathological changes of liver fibrosis, as well as through downregulation of TGF-β1 and phosphorylated SMAD3 both in vitro and in vivo.ConclusionsThese findings suggested that ADSC transplantation can ameliorate insulin resistance, hyperglycemia, and liver fibrosis via suppressing TGF-β1/SMAD3 signaling, which may provide a potential treatment strategy for liver fibrosis of T2D.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0743-7) contains supplementary material, which is available to authorized users.

CRISPR/Cas9-mediated gene therapy holds great promise for the treatment of human diseases. The protospacer adjacent motif (PAM), the sequence adjacent to the target sequence, is an essential targeting component for the design of CRISPR/Cas9-mediated gene editing. However, currently, very few studies have attempted to directly study the PAM sequence in human cells. To address this issue, the authors develop a dual fluorescence reporter system that could be harnessed for identifying functional PAMs for genome editing endonuclease, including Cas9. With this system, the authors investigate the effects of different PAM sequences for SaCas9, which is small and has the advantage of allowing in vivo genome editing, and found only 5'-NNGRRT-3' PAM could induced sufficient target cleavage with multi-sites. The authors also found SaCas9 possesses higher activity than SpCas9 or FnCpf1 via plasmids (episomal) and chromosomes with integrated eGFP-based comparison. Taken together, the authors show that a dual fluorescence reporter system is a means to identifying a functional PAM and quantitatively comparing the efficiency of different genome editing endonucleases with the similar or identical target sequence in human cells.

CRISPR-Staphylococcus aureus Cas9 (CRISPR-SaCas9) has been harnessed as an effective in vivo genome-editing tool to manipulate genomes. However, off-target effects remain a major bottleneck that precludes safe and reliable applications in genome editing. Here, we characterize the off-target effects of wild-type (WT) SaCas9 at single-nucleotide (single-nt) resolution and describe a directional screening system to identify novel SaCas9 variants with desired properties in human cells. Using this system, we identified enhanced-fidelity SaCas9 (efSaCas9) (variant Mut268 harboring the single mutation of N260D), which could effectively distinguish and reject single base-pair mismatches. We demonstrate dramatically reduced off-target effects (approximately 2-to 93-fold improvements) of Mut268 compared to WT using targeted deep-sequencing analyses. To understand the structural origin of the fidelity enhancement, we find that N260, located in the REC3 domain, orchestrates an extensive network of contacts between REC3 and the guide RNA-DNA heteroduplex. efSa-Cas9 can be broadly used in genome-editing applications that require high fidelity. Furthermore, this study provides a general strategy to rapidly evolve other desired CRISPR-Cas9 traits besides enhanced fidelity, to expand the utility of the CRISPR toolkit.

Cre-loxP, as one of the site-specific genetic manipulation tools, offers a method to study the spatial and temporal regulation of gene expression/inactivation in order to decipher gene function. CRISPR/Cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research. Whether the traditional site-specific genetic manipulation tool and CRISPR/Cas9 could be combined to create a novel genetic tool for highly specific gene editing is not clear. Here, we successfully generated a CRISPR/Cas9-loxP system to perform gene editing in human cells, providing the proof of principle that these two technologies can be used together for the first time. We also showed that distinct non-homologous end-joining (NHEJ) patterns from CRISPR/Cas9-mediated gene editing of the targeting sequence locates at the level of plasmids (episomal) and chromosomes. Specially, the CRISPR/Cas9-mediated NHEJ pattern in the nuclear genome favors deletions (64%–68% at the human AAVS1 locus versus 4%–28% plasmid DNA). CRISPR/Cas9-loxP, a novel site-specific genetic manipulation tool, offers a platform for the dissection of gene function and molecular insights into DNA-repair pathways.

Clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) of bacterial adaptive immunity have been adopted as a powerful and versatile tool for manipulation of the genome. This paradigm has been widely applied in biological research and treatments of animal or cellular disease models. A critical feature of CRISPR-Cas is the protospacer adjacent motif (PAM), which dictates the DNA target recognition mechanism of Cas proteins. While, direct identifying functional PAM sequences in human cells remains a challenge. Here, we developed a positive screen system termed PAM-DOSE (PAM Definition by Observable Sequence Excision) to delineate the functional PAMs in human cells. Specifically, the PAM libraries for CRISPR-Cas (SpCas9, SpCas9-NG, FnCas12a, AsCas12a, LbCas12a and MbCas12a) were generated and the corresponding CRISPR-Cas mediated cleaved fragments with functional PAM in human cells were harvested for DNA sequencing, which could be tracked and visualized with either florescence microscopy or flow cytometry analysis. With this system, we identified the functional PAMs of CRISPR-Cas members. We also found that spacer sequence affects the PAM preference of Cas proteins. This method will facilitate identification of functional PAMs for Cas-mediated human genome editing applications.

Genome editing using RNA‐guided nucleases in their ribonucleoprotein (RNP) form represents a promising strategy for gene modification and therapy because they are free of exogenous DNA integration and have reduced toxicity in vivo and ex vivo. However, genome editing by Cas9 nuclease from Staphylococcus aureus (SaCas9) has not been reported in its RNP form, which recognizes a longer protospacer adjacent motif (PAM), 5′‐NNGRRT‐3′, compared with Streptococcus pyogenes Cas9 (SpCas9) of 5′‐NGG‐3′ PAM. Here, SaCas9‐RNP‐mediated genome editing is reported in human cells. The SaCas9‐RNP displayed efficient genome editing activities of enhanced green fluorescent protein (EGFP) coding gene as well as three endogenous genes (OPA1, RS1, and VEGFA). Further, SaCas9‐RNP is successfully implemented to correct a pathogenic RS1 mutation for X‐linked juvenile retinoschisis. It is also shown that off‐target effects triggered by SaCas9‐RNP are undetectable by targeted deep sequencing. Collectively, this study demonstrates the potential of SaCas9‐RNP‐mediated genome editing in human cells, which could facilitate genome‐editing‐based therapy.

BackgroundX-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disorder caused by mutations in the ABCD1 gene. Approximately 20% of X-ALD female carriers may develop neurological symptoms. Skewed X chromosome inactivation (XCI) has been proposed to influence the manifestation of symptoms in X-ALD carriers, but data remain conflicting so far. We identified a three generation kindred, with five heterozygous females, including two manifesting carriers. XCI pattern and the ABCD1 allele expression were assessed in order to determine if symptoms in X-ALD carriers could be related to skewed XCI and whether skewing within this family is more consistent with genetically influenced or completely random XCI.ResultsWe found a high frequency of skewing in this family. Four of five females had skewed XCI, including two manifesting carriers favoring the mutant allele, one asymptomatic carrier favoring the normal allele, and one female who was not an X-ALD carrier. Known causes of skewing, such as chromosomal abnormalities, selection against deleterious alleles, XIST promoter mutations, were not consistent with our results.ConclusionsOur data support that skewed XCI in favor of the mutant ABCD1 allele would be associated with the manifestation of heterozygous symptoms. Furthermore, XCI skewing in this family is genetically influenced. However, the underlying mechanism remains to be substantiated by further experiments.