NmeCas9 is an intrinsically high-fidelity genome-editing platform

Background

The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery.

Results

Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5′-N₄GATT-3′), for NmeCas9 genome editing in human cells.

Conclusions

Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.

     

Protective effects of mitochondrion-targeted peptide SS-31 against hind limb ischemia-reperfusion injury

Hind limb ischemia-reperfusion injury is an important pathology in vascular surgery. Reactive oxygen species are thought to be involved in the pathogenesis of hind limb ischemia-reperfusion injury. SS-31, which belongs to a family of mitochondrion-targeted peptide antioxidants, was shown to reduce mitochondrial reactive oxygen species production. In this study, we investigated whether the treatment of SS-31 could protect hind limb from ischemia-reperfusion injury in a mouse model. The results showed that SS-31 treatment either before or after ischemia exhibited similar protective effects. Histopathologically, SS-31 treatment prevented the IR-induced histological deterioration compared with the corresponding vehicle control. SS-31 treatment diminished oxidative stress revealed by the reduced malondialdehyde level and increased activities and protein levels of Sod and catalase. Cellular ATP contents and mitochondrial membrane potential increased and the level of cytosolic cytC was decreased after SS-31 treatment in this IR model, demonstrating that mitochondria were protected. The IR-induced increase of levels of inflammatory factors, such as Tnf-α and Il-1β, was prevented by SS-31 treatment. In agreement with the reduced cytosolic cytC, cleaved-caspase 3 was kept at a very low level after SS-31 treatment. Overall, the effect of SS-31 treatment before ischemia is mildly more effective than that after ischemia. In conclusion, our results demonstrate that SS-31 confers a protective effect in the mouse model of hind limb ischemia-reperfusion injury preventatively and therapeutically.     

Combinatorial Control of Recruitment of a Variant PRC1.6 Complex in Embryonic Stem Cells

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Epr detection of endogenous nitric oxide in postischemic heart using lipid and aqueous-soluble dithiocarbamate-iron complexes

Spin-trapping techniques combined with electron paramagnetic resonance (EPR) spectroscopy to measure nitric oxide (·NO) production were compared in the ischemic-reperfused myocardium for the first time, using both aqueous-soluble and lipophilic complexes of reduced iron (Fe) with dithiocarbamate derivatives. The aqueous-soluble complex of Fe and N-methyl-D-glucamine dithiocarbamate (MGD) formed MGD2-Fe-NO complex with a characteristic triplet EPR signal (a_N12.5 G and g_iso = 2.04) at room temperature, in native isolated rat hearts following 40 min global ischemia and 15 min reperfusion. Diethyldithiocarbamate (DETC) and Fe formed in ischemic-reperfused myocardium the lipophilic DETC2-Fe-NO complex exhibiting an EPR signal (g = 2.04 and g = 2.02 at 77K) with a triplet hyperfine structure at g. Dithiocarbamate-Fe-NO complexes detected by both trapping agents were abolished by the ·NO synthase inhibitor, N^G-nitro-L-arginine methyl ester. Quantitatively, both trapping procedures provi ded similar values for tissue ·NO production, which were observed primarily during ischemia. Postischemic hemodynamic recovery of the heart was not affected by the trapping procedure. (Mol Cell Biochem 175: 91–97, 1997)     

Activation of the neutrophil and loss of plasma glutathione during Mg-deficiency – modulation by nitric oxide synthase inhibition

Sprague-Dawley rats (200 g) were fed either a Mg-deficient or Mg-sufficient diet for 3 weeks. An enriched neutrophil fraction (>85%) was isolated from the blood by sodium metrizoate/dextran gradient centrifugation. Using the superoxide dismutase (SOD)-inhibitable cytochrome c reduction assay, the basal activity of neutrophils isolated from the Mg-deficient rats were found elevated 5 fold after two weeks, and up to 7 fold after three weeks on the diet. Upon challenge by phorbol myristate acetate (PMA), unlike the Mg-sufficient cells, the Mg-deficient cells exhibited no significant activation. Treatment of the Mg-deficient rats with the nitric oxide (NO)-synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) in the drinking water, significantly attenuated the basal superoxide producing activity of the neutrophils and partially restored its response to PMA challenge. In association with the neutrophil activation. Mg-deficiency resulted in 70% decrease in plasma glutathione and 220% increase in Fe-promoted, thiobarbituric acid reactive substance (TBARS) levels; both changes were significantly attenuated by L-NAME treatment. The results suggest that neutrophils from Mg-deficient rats are activated endogenously to generate oxy-radicals which might directly mediate the in vivo peroxidative indices during Mg-deficiency. Furthermore, the neutrophil activity was lowered by NO-synthase inhibition suggesting that NO overproduction during Mg-deficiency participates in the neutrophil activation process.     

光敏核不育水稻农垦58S 41 kD蛋白质的N—端序列分析

光敏核不育性受1对或2对或3对隐性主基因控制,这反应了光敏核不育特性遗传机制的复杂性。张端品等用形态标记法将农垦58S光敏核不育基因定位于第5染色体。胡学应和万邦惠用同工酶法以农垦58S/02428 F_2群体为材料,将光敏核不育基因定位于第6和11染色体;Zhang等用RFLP法以32001S/明恢63 F_2群体为材料将不育基因定位于第3和7染色体。这三种方法所得到的定位结果完全不同,综合起来,第3、5、6、7和11染色体均有光敏核不育基因的位点,由此结果可得出两种解释:1.光敏核不育性由多对基因、至少5对基因控制;2.上述定位方法均是以不育(可育)性状在F_2群体中的分离模式为依据,育性划分界线的不同将会造成分离群体中单株表现值的差异,从而影响定位结果的精确性;再者不同实验室使用的材料不一致,已知不同遗传背景和光温条件影响光敏核不育基因的表达。因此,染色体定位结果有待确证。光敏核不育基因在染色体上定位的复杂性和不一致在某种程度上影响了基因克隆和光敏核不育分子机制的研究。无论光敏核不育性的遗传机制如何复杂,上述结果     

中国人β神经生长因子前体基因的克隆及其序列分析

从正常人外周血白细胞中提取基因组ＤＮＡ,用ＰＣＲ扩增神经生长因子（ＮＧＦ）β亚基前体的全长编码区序列,将其克隆到Ｔ－ｖｅｃｔｏｒ（原始质粒为ｐＢｌｕｅｓｃｒｉｐｔⅡＳＫ（＋））上,取两个独立的克隆采用自动测序仪进行双链ＤＮＡ双向测序,结果表明：两个克隆的序列完全相同,该序列与国外报道的ＮＧＦ序列有一个碱基的差别,从而导致ＮＧＦ前导肽中一个氨基酸的改变,而成熟的ＮＧＦ序列没有改变。     

Monitoring single-cell pharmacokinetics by capillary electrophoresis and laser-induced native fluorescence

The quantification of insulin released from single cells of the insulin-secreting cell line βTC3 permeabilized by digitonin is demonstrated. A simple method for monitoring the on-column release process by using capillary electrophoresis and laser-induced native fluorescence detection is described. Quantitative measurements of both the amount of insulin released and the amount remaining in the cell can be achieved simultaneously. This protocol provides an alternative approach to the study of cell secretion in the fields of neuroscience and endocrinology.