抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.

/pdf/genome-engineering-using-the-crispr-cas9-system-4x8nw03mgu.pdf

Genome engineering using the CRISPR-Cas9 system

The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

The new frontier of genome engineering with CRISPR-Cas9

http://knockout.cwru.edu/publications/hsucrisprreview.pdf

Development and applications of CRISPR-Cas9 for genome engineering.

https://limlab.ucsf.edu/papers/pdfs/lsq_2013.pdf

Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.

LbCpf1, derived from Lachnospiraceae bacterium ND2006, is a CRISPR RNA-guided endonuclease and holds promise for therapeutic applications. Here we show that LbCpf1 can be used for therapeutic gene editing in a mouse model of age-related macular degeneration (AMD). The intravitreal delivery of LbCpf1, targeted to two angiogenesis-associated genes encoding vascular endothelial growth factor A (Vegfa) and hypoxia inducing factor 1a (Hif1a), using adeno-associated virus, led to efficient gene disruption with no apparent off-target effects in the retina and retinal pigment epithelium (RPE) cells. Importantly, LbCpf1 targeted to Vegfa or Hif1a in RPE cells reduced the area of laser-induced choroidal neovascularization as efficiently as aflibercept, an anti-VEGF drug currently used in the clinic, without inducing cone dysfunction. Unlike aflibercept, LbCpf1 targeted to Vegfa or Hif1a achieved a long-term therapeutic effect on CNV, potentially avoiding repetitive injections. Taken together, these results indicate that LbCpf1-mediated in vivo genome editing to ablate pathologic angiogenesis provides an effective strategy for the treatment of AMD and other neovascularization-associated diseases.

/pdf/crispr-lbcpf1-prevents-choroidal-neovascularization-in-a-222jwzw9b9.pdf

CRISPR-LbCpf1 prevents choroidal neovascularization in a mouse model of age-related macular degeneration

Functional Rescue of Dystrophin Deficiency in Mice Caused by Frameshift Mutations Using Campylobacter jejuni Cas9.

VOLUME 34 NUMBER 10 OCTOBER 2016 NATURE BIOTECHNOLOGY To the Editor: The US Department of Agriculture (USDA) recently announced that regulation of a CRISPR-Cas9 gene-edited mushroom, in which a polyphenol oxidase (PPO) gene had been mutated to avoid (or delay) browning, fell outside of genetically modified organism (GMO) legislation1. However, we remain concerned that the approved mushroom may still contain tiny fragments of foreign DNA in its genome. If foreign DNA is present after CRISPR-Cas9 editing, regulatory approval would be required under current GMO legislation. In a letter (available in ref. 1 and at https://www.aphis.usda.gov/aphis/home/) submitted to the USDA, Yinong Yang, a researcher at Pennsylvania State University, outlined how the gene-edited mushroom was produced. Mushroom protoplasts (fungal cells with no cell wall) were transfected with plasmids encoding Cas9 and a guide RNA (gRNA) specific for a PPO gene. Transfected protoplasts were regenerated to produce mushrooms that contained small deletions (1–14 base pairs (bp)) in the PPO gene. The researchers analyzed the genome of edited fungi using PCR and Southern blot analysis and found no evidence of foreign DNA in the genome. In response, the USDA decided that the gene-edited mushroom fell outside of GMO regulations and that the US government had no authority to regulate this product. Melbourne, Victoria, Australia. 19Division of Surgical Oncology, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 20Molecular Genomics, Research Division, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 21Bioinformatics and Cancer Genomics, Research Division, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 22The Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 23Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 24The Department of Surgery, the University of Melbourne, Parkville, Victoria, Australia. 25The Jack Brockhoff Familial Cancer Centre, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 26Hereditary Cancer Program, BC Cancer Agency, Vancouver, Canada. 27Molecular Biomarkers and Translational Genomics Laboratory, Research Department, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 28Department of Nursing, the Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 29Department of Pathology, the Royal Women’s Hospital, Parkville, Victoria, Australia. 30Department of Pathology, University of Dammam, Dammam, Saudi Arabia. 31Department of Forensic Medicine, School of Public Health and Preventive Medicine, Monash University, Victoria, Australia. 32The Garvan Institute, Sydney, New South Wales, Australia. Correspondence should be addressed to: d.bowtell@petermac.org

Bypassing GMO regulations with CRISPR gene editing

DddA-derived cytosine base editors (DdCBEs), composed of the split interbacterial toxin DddAtox, transcription activator-like effector (TALE), and uracil glycosylase inhibitor (UGI), enable targeted C-to-T base conversions in mitochondrial DNA (mtDNA). Here, we demonstrate highly efficient mtDNA editing in mouse embryos using custom-designed DdCBEs. We target the mitochondrial gene, MT-ND5 (ND5), which encodes a subunit of NADH dehydrogenase that catalyzes NADH dehydration and electron transfer to ubiquinone, to obtain several mtDNA mutations, including m.G12918A associated with human mitochondrial diseases and m.C12336T that incorporates a premature stop codon, creating mitochondrial disease models in mice and demonstrating a potential for the treatment of mitochondrial disorders. Split DddA-derived base editors fused to TALEs enable mitochondrial DNA editing. Here the authors demonstrate their use in mouse embryos with germline transmission.

/pdf/mitochondrial-dna-editing-in-mice-with-ddda-tale-fusion-2yuv5dwwnq.pdf

Jin-Soo Kim

Papers

CRISPR-LbCpf1 prevents choroidal neovascularization in a mouse model of age-related macular degeneration

Functional Rescue of Dystrophin Deficiency in Mice Caused by Frameshift Mutations Using Campylobacter jejuni Cas9.

Bypassing GMO regulations with CRISPR gene editing

Mitochondrial DNA editing in mice with DddA-TALE fusion deaminases

Growth of Room-Temperature 1.32-mum-Emitting InAs Quantum Dots Using GaAs/InAs Quasi-Monolayers