Journal: Scientific Reports

Article Title: Targeted mutagenesis in soybean using the CRISPR-Cas9 system

doi: 10.1038/srep10342

Figure Lengend Snippet: Construction of binary vectors for genome editing in soybean. Cas9 fused with a single nuclear localization signal (NLS) is expressed with a Cauliflower mosaic virus 35s (CaMV 35s) promoter. Synthetic guide RNA (sgRNA) is derived using U6 promoters. ( a ) Arabidopsis thaliana U6-26 promoter ( b ) Glycine max U6-10 promoter. Sequences containing two Bsa I sites are located between the U6 promoter and the sgRNA scaffold. These sequences can be easily replaced with a gene-specific sgRNA seed. LB: left border; RB: right border.

Article Snippet: Arabidopsis U6-26 and soybean U6-10 promoters with sgRNA were synthesized (Genscript, Nanjing, China) ( and ) and cloned into pUC57-Kan vectors to generate pUC57-AtU6-26-sgRNA and pUC57-GmU6-10-sgRNA plasmids, respectively.

Techniques: Virus, Derivative Assay

Journal: Nucleic Acids Research

Article Title: Short tandem repeat stutter model inferred from direct measurement of in vitro stutter noise

doi: 10.1093/nar/gky1318

Figure Lengend Snippet: The synthetic STR experiment summary. ( A ) Schematic description of the synthetic library. In each plasmid, a different synthetic STR construct was designed, synthesized and clone-sequenced for various STR types and length. The STR was designed within a context of an Illumina Truseq-HT dual index library to enable for nested PCR amplification at two time points (T 2 - amplification using outer primers only, T 3 -amplification using inner primers followed amplification by outer primers). The library is flanked by BsrDI restriction sites to enable direct sequencing of the STR library without amplification (T 1 ). Internal barcode (yellow triangle) is a short sequence, unique to each STR length to detect for cross-contamination. See text and methods for elaboration and for the designed constructs. ( B ) AC STRs repeat-number histograms, as were interpreted from sequencing results (T 1 , T 2 and T 3 ), compared to their expected length, T 0 (designed sequence). ( C ) Sequencing analysis results of each STR type, repeat-number and time point described as the percentage of the original (designed) signal from all the reads. Dashed line at the 5% marks the lower threshold of analysis: data points below the mark were deemed too noisy and were excluded from downstream analysis.

Article Snippet: STR plasmid design: Sequence verified cloned plasmids containing synthetic STRs of different types and sizes ( ) were ordered from either IDT or GenScript (pIDT-kan and modified puc57-Kan vectors, respectively).

Techniques: Plasmid Preparation, Construct, Synthesized, Nested PCR, Amplification, Sequencing

Journal: Science Advances

Article Title: Versatile transgenic multistage effector-gene combinations for Plasmodium falciparum suppression in Anopheles

doi: 10.1126/sciadv.aay5898

Figure Lengend Snippet: ( A ) Schematic illustration of transgenic targeting of parasite sporozoite infection stages and the design of AsVg -driven transgenes to be expressed in the fatbody after the blood meal, to specifically target the parasites at this stage. ( B ) Schematic representation of single-chain antibody (ScFv) targeting the CSP protein fused to AMP. The single-chain antibodies consist of variable regions of the V H heavy and V L light chains. Each transgene encodes a short 5–amino acid polypeptide linker between V H and V L and a long 15–amino acid (aa) sequence linking the V H to the AMP peptides (CecC, PLA2, and Scorpine), including the CecA signal peptide sequence (SP). Three individual transformation plasmids, pBAC-AsVg-CecC (or PLA2 , Scorpine )– ScFv (with the red fluorescent eye reporter gene 3xP3 dsRed ), were used for the germline transformation. AsVg promoter with the same AsVg endogenous terminator ( AsVg 3′-UTR ) was used. ( C ) Fluorescent images of a positive larva and an adult transgenic ScorpScFv mosquito. ( D ) PCR validation of the partial transgene cassette (~500 bp) of CecScFv , PLAScFv , and ScorpScFv in the transgenic mosquitoes. ( E ) Transcript abundance of the transgene in the fatbody of ScorpScFv transgenic line at various time points PBM. Each bar represents the relative fold change in the transgene as compared to the control at time 0 hour. The S7 ribosomal gene was used to normalize the cDNA templates. Error bars indicate SEM. ( F to I ) P. falciparum (NF54) oocyst and sporozoite infection intensities and prevalence of the three transgenic ScFv lines (PLAScFv, CecScFv, and ScorpScFv) at 8 dpi in the gut or 14 dpi in the salivary glands (SG) without (F and G) or with (H and I) additional naïve blood meals at days 5 and 9 post-infectious blood meal (PIBM). Each dot represents the number of parasites in an individual midgut or salivary glands, and the horizontal lines (red) indicate the median values. Detailed statistical analysis is presented in table S2.

Article Snippet: The transgene cassette, including the AsVg promoter, CecScFv , and AsVg 3′- UTR and terminator sequences, was codon-optimized (for ScFv-CSP), synthesized through GenScript Inc. (Supplementary Materials), and cloned into the pUC57-Kan cloning vector.

Techniques: Transgenic Assay, Infection, Sequencing, Transformation Assay, Biomarker Discovery, Control