Scheme of classifying off-target cleavage sites with the sgRNAcas9 program.

<p>(A) Different segments of the target sequence. (B) The potential off-target cleavage sites (POT) were classified into three categories by count number and position of mismatches. (C) Distinction of true and false off-targets. (D) Evaluating the degree of risk of the POT. The dangerous degree of POT from low to high is dependent on the number and position of mismatches.</p

Example of CRISPR on-target sequences found by sgRNAcas9.

<p>(A) The paired-gRNA target sites of <i>Emx1</i> gene found by sgRNAcas9 program and the paired-gRNA searching mode. (B) Illustration of one pair sgRNA targeting at exon1 of <i>Emx1</i>.</p

Running time for finding CRISPR target sites and searching off-targets.

<p>Note: Default parameter is used to perform different searching mode by sgRNAcas9.pl. Length of exon: exon 1 (898 bp), exon 2 (185 bp), and exon 3 (1105 bp).</p

Example of the classification of potential off-target sequences by sgRNAcas9.

<p>(A) Searching off-target sites for each sgRNA targeting at human <i>Emx1</i> (hEMX1) gene. (B) Classifying POT by number and position of mismatches into three types. Notes: “seed_ident”, strand for seed region, was to identity to on-target sites (Type I). “region I_ident”, strand for region I identical to on-target site (Type II). “random_0_3M”, strand for regions with 1∼3 mismatched bases randomly distributed on the region I, II and III, but with at least one mismatched base located on the region I (Type III).</p

Workflow diagram of sgRNAcas9 software.

<p>Finding CRISPR target sites and off-target risk assessment includes 4 basic steps. It is noteworthy that to evaluate off-target effects exaclty, different types of potential off-target cleavage sites (POT) are classified.</p

Additional file 1 of CRISPR/Cas9-mediated knockout of intracellular molecule SHP-1 enhances tumor-killing ability of CD133-targeted CAR T cells in vitro

Supplementary Material

Heritable Multiplex Genetic Engineering in Rats Using CRISPR/Cas9

<div><p>The CRISPR/Cas9 system has been proven to be an efficient gene-editing tool for genome modification of cells and organisms. Multiplex genetic engineering in rat holds a bright future for the study of complex disease. Here, we show that this system enables the simultaneous disruption of four genes (<i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i>) in rats in one-step, by co-injection of Cas9 mRNA and sgRNAs into fertilized eggs. We further observed the gene modifications are germline transmittable, and confirmed the off-target mutagenesis and mosaicism are rarely detected by comprehensive analysis. Thus, the CRISPR/Cas9 system makes it possible to efficiently and reliably generate gene knock-out rats.</p></div

Putative off-target sites of Ar-A and Ar-B sgRNAs.

<p>Putative off-target sites of Ar-A and Ar-B sgRNAs.</p

The sgRNA:Cas9-mediated modifications in Cas9 expression oocytes.

<p>(A and E) Schematic of two sgRNAs targeting the mouse <i>Ar</i> gene and NLRP3 gene. (B) PCR products of the targeted, and detection of Cas9-mediated on-target cleavage on wild-type zygotes by co-injection Cas9 mRNA and Ar sgRNAs by T7EI cleavage assay. (C and F) PCR products of the targeted, and detection of Cas9-mediated on-target cleavage on Zp3-Cas9 transgenic mouse zygotes injected with sgRNA only by T7EI cleavage assay. (D and G) Sequencing results of modified alleles in Zp3-Cas9 transgenic mouse zygotes injected with sgRNAs.</p

Summary of the mutation of the founder rats.

<p>Single sgRNA/Gene: A mixture of 4 sgRNAs, each targeting a single site in each of the 4 genes. Dual sgRNAs/Gene: Another distinct sgRNA of each gene, together with the tested 4 sgRNAs targeting double site in each of the 4 genes.</p