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.

The combined availability of whole genome sequences and genome editing tools is set to revolutionize the field of fruit biotechnology by enabling the introduction of targeted genetic changes with unprecedented control and accuracy, both to explore emergent phenotypes and to introduce new functionalities. Although plasmid-mediated delivery of genome editing components to plant cells is very efficient, it also presents some drawbacks, such as possible random integration of plasmid sequences in the host genome. Additionally, it may well be intercepted by current process-based GMO regulations, complicating the path to commercialization of improved varieties. Here, we explore direct delivery of purified CRISPR/Cas9 ribonucleoproteins (RNPs) to the protoplast of grape cultivar Chardonnay and apple cultivar such as Golden delicious fruit crop plants for efficient targeted mutagenesis. We targeted MLO-7, a susceptible gene in order to increase resistance to powdery mildew in grape cultivar and DIPM-1, DIPM-2, and DIPM-4 in the apple to increase resistance to fire blight disease. Furthermore, efficient protoplast transformation, the molar ratio of Cas9 and sgRNAs were optimized for each grape and apple cultivar. The targeted mutagenesis insertion and deletion rate was analyzed using targeted deep sequencing. Our results demonstrate that direct delivery of CRISPR/Cas9 RNPs to the protoplast system enables targeted gene editing and paves the way to the generation of DNA-free genome edited grapevine and apple plants.

/pdf/dna-free-genetically-edited-grapevine-and-apple-protoplast-13gqdgudqk.pdf

DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins

A collection of TALENs targeted to 18,740 human protein-coding genes will facilitate genetic engineering of human cells.

A library of TAL effector nucleases spanning the human genome

Cpf1, a type V CRISPR effector, recognizes a thymidine-rich protospacer-adjacent motif and induces cohesive double-stranded breaks at the target site guided by a single CRISPR RNA (crRNA). Here we show that Cpf1 can be used as a tool for DNA-free editing of plant genomes. We describe the delivery of recombinant Cpf1 proteins with in vitro transcribed or chemically synthesized target-specific crRNAs into protoplasts isolated from soybean and wild tobacco. Designed crRNAs are unique and do not have similar sequences (≤3 mismatches) in the entire soybean reference genome. Targeted deep sequencing analyses show that mutations are successfully induced in FAD2 paralogues in soybean and AOC in wild tobacco. Unlike SpCas9, Cpf1 mainly induces various nucleotide deletions at target sites. No significant mutations are detected at potential off-target sites in the soybean genome. These results demonstrate that Cpf1–crRNA complex is an effective DNA-free genome-editing tool for plant genome editing. Cpf1 is a type V CRISPR effector protein that has different target and guide RNA requirements compared to Cas9, thus offering an addition tool for precision genome editing. Here Kimet al. show that Cpf1 ribonucleoprotein can be introduced into protoplasts and used for transgene-free gene editing in plants.

/pdf/crispr-cpf1-mediated-dna-free-plant-genome-editing-s1ycqzvnhg.pdf

CRISPR/Cpf1-mediated DNA-free plant genome editing

Knockout mice created by TALEN-mediated gene targeting

To the Editor: Programmable nucleases such as Cas9 RNA-guided engineered nucleases (RGENs)1 enable gene knockout in cultured cells and organisms by producing site-specific DNA double-strand breaks, whose repair via error-prone nonhomologous end joining gives rise to small insertions and deletions (indels) at target sites, often causing frameshift mutations in a protein-coding sequence2. The efficiency of this method can be reduced by in-frame mutations via microhomology-mediated end joining3,4 (Fig. 1a). Here we present a computer program that assists in the choice of Cas9 nuclease, zinc-finger nuclease and transcription activator–like effector nuclease (TALEN) target sites, using microhomology prediction to achieve efficient gene disruption in cell lines and whole organisms. First we examined the mutations induced by ten TALENs and ten RGENs in human cells via deep sequencing (Supplementary Table 1 and Supplementary Methods). We focused our analysis on deletions because they are much more prevalent than insertions (98.7% vs. 1.3%, respectively, for TALENs; 75.1% vs. 24.9% for RGENs) and because microhomology is irrelevant for insertions. In aggregate, microhomologies of 2–8 bases were found in 44.3% and 52.7% of all deletions induced by TALENs and RGENs, respectively (Supplementary Fig. 1 and Supplementary Table 2). Thus, 43.7% (0.987 × 0.443) and 39.6% (0.751 × 0.527) of all the mutations induced by TALENs and RGENs, respectively, were associated with microhomology. At a given nuclease target site, the effect of these microhomologyassociated deletions can be predicted. In the extreme cases, (i) all deletions cause frameshifts in a protein-coding gene or (ii) no deletions cause frameshifts. In contrast, one-third of microhomologyindependent deletions result in in-frame mutations. Assuming that ~60% of indels are microhomology independent on average, the fraction of in-frame mutations at a given site can range from 20% (60%/3 + 0%) to 60% (60%/3 + 40%), a threefold difference between the two extreme cases. Because most eukaryotic cells are diploid rather than haploid, the fraction of null cells carrying two outof-frame mutations can range from 16% (0.40 × 0.40) to 64% (0.80 × 0.80), depending on the choice of target site. A careful analysis of indel sequences also revealed that the frequency of microhomology-associated deletions depends on both the size of the microhomology and the length of the deletion (Supplementary Fig. 2). On the basis of these observations, we developed a simple formula and a computer program (Supplementary Fig. 3) to predict the deletion patterns at a given nuclease target site that are associated with microhomology of at least two bases (Fig. 1b and Supplementary Note). We assigned a pattern score to each deletion pattern and a microhomology score (equaling the sum of pattern scores) to each target site. We then obtained an out-of-frame score at a given site by dividing the sum of pattern scores assigned to frameshifting deletions by the microhomology score. To evaluate the utility of our scoring system, we arbitrarily chose two target sites in exons, one with a high score (top 20%) and the other with a low score (bottom 20%) in each of nine human genes. We targeted a total of 6 and 12 sites in human cells with RGENs and TALENs, respectively (Supplementary Table 3), and then analyzed the mutant patterns by deep sequencing (Supplementary Table 4). High-score sites produced out-of-frame indels much more frequently than did low-score sites in all nine pairs (Fig. 1c), even at two adjacent target sites separated by 29 bases in the MCM6 gene (Supplementary Fig. 4). On average, the high-score sites and low-score sites produced frameshifting indels at frequencies of 79.3% and 42.5%, respectively (Student’s t-test, P < 0.01). We then tested in HeLa cells 68 new RGENs that target different genes (Supplementary Table 5). Again, out-of-frame scores correlated well with the frequencies of frameshifting indels or deletions (Pearson coefficient = 0.717 and 0.732, respectively) (Fig. 1d and Supplementary Fig. 5). The frequencies of out-of-frame indels ranged from 38.7% to 94.0%. In a diploid human cell, the probability of obtaining null clones would thus range from 15.0% (0.387 × 0.387) to 88.4%. Most cancer cell lines including HeLa are multi-ploid (>3n), making it even more important to choose high-score sites. We expect that the scoring system would work even better for TALENs because TALENs induce microhomology-independent insertions much less frequently than do RGENs (Supplementary Fig. 1b). We also analyzed the genotypes of 81 mice carrying mutations produced via TALENs5 or RGENs6, from our previous studies. The frequencies of out-of-frame deletions correlated well with predicted scores (Pearson coefficient = 0.996; Supplementary Fig. 6). In summary, we developed a scoring system to estimate the frequency of microhomology-associated deletions at nuclease

Jin-Soo Kim

Papers

DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins

A library of TAL effector nucleases spanning the human genome

CRISPR/Cpf1-mediated DNA-free plant genome editing

Knockout mice created by TALEN-mediated gene targeting

Microhomology-based choice of Cas9 nuclease target sites