Sangsu Bae

Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases

RNA-guided endonucleases (RGENs), derived from the prokaryotic adaptive immune system known as CRISPR/Cas, enable targeted genome engineering in cells and organisms. RGENs are ribonucleoproteins that consist of guide RNA and Cas9, a protein component originated from Streptococcus pyogenes. These enzymes cleave chromosomal DNA, whose sequence is complementary, to guide RNA in a targeted manner, producing site-specific DNA double-strand breaks (DSBs), the repair of which gives rise to targeted genome modifications. Despite broad interest in RGEN-mediated genome editing, these nucleases are limited by off-target mutations and unwanted chromosomal translocations associated with off-target DNA cleavages. Here, we show that off-target effects of RGENs can be reduced below the detection limits of deep sequencing by choosing unique target sequences in the genome and modifying both guide RNA and Cas9. We found that both the composition and structure of guide RNA can affect RGEN activities in cells to reduce off-target effects. RGENs efficiently discriminated on-target sites from off-target sites that differ by two bases. Furthermore, exome sequencing analysis showed that no off-target mutations were induced by two RGENs in four clonal populations of mutant cells. In addition, paired Cas9 nickases, composed of D10A Cas9 and guide RNA, which generate two single-strand breaks (SSBs) or nicks on different DNA strands, were highly specific in human cells, avoiding off-target mutations without sacrificing genome-editing efficiency. Interestingly, paired nickases induced chromosomal deletions in a targeted manner without causing unwanted translocations. Our results highlight the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to avoid or reduce RGEN-induced off-target mutations.

Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases

Summary: The Type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is an adaptive immune response in prokaryotes, protecting host cells against invading phages or plasmids by cleaving these foreign DNA species in a targeted manner. CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) enable genome editing in cultured cells, animals and plants, but are limited by off-target mutations. Here, we present a novel algorithm termed Cas-OFFinder that searches for potential off-target sites in a given genome or user-defined sequences. Unlike other algorithms currently available for identification of RGEN off-target sites, Cas-OFFinder is not limited by the number of mismatches and allows variations in protospacer-adjacent motif sequences recognized by Cas9, the essential protein component in RGENs. Cas-OFFinder is available as a command-line program or accessible via our website. Availability and implementation: Cas-OFFinder free access at http://www.rgenome.net/cas-offinder. Contact: baesau@snu.ac.kr or jskim01@snu.ac.kr

Microhomology-based choice of Cas9 nuclease target sites

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

Functional Correction of Large Factor VIII Gene Chromosomal Inversions in Hemophilia A Patient-Derived iPSCs Using CRISPR-Cas9

Hemophilia A is an X-linked genetic disorder caused by mutations in the F8 gene, which encodes the blood coagulation factor VIII. Almost half of all severe hemophilia A cases result from two gross (140-kbp or 600-kbp) chromosomal inversions that involve introns 1 and 22 of the F8 gene, respectively. We derived induced pluripotent stem cells (iPSCs) from patients with these inversion genotypes and used CRISPR-Cas9 nucleases to revert these chromosomal segments back to the WT situation. We isolated inversion-corrected iPSCs with frequencies of up to 6.7% without detectable off-target mutations based on whole-genome sequencing or targeted deep sequencing. Endothelial cells differentiated from corrected iPSCs expressed the F8 gene and functionally rescued factor VIII deficiency in an otherwise lethal mouse model of hemophilia. Our results therefore provide a proof of principle for functional correction of large chromosomal rearrangements in patient-derived iPSCs and suggest potential therapeutic applications.

DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins

Microalgae are versatile organisms capable of converting CO2, H2O, and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity, and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity.

Intrinsic Z-DNA Is Stabilized by the Conformational Selection Mechanism of Z-DNA-Binding Proteins

Z-DNA, a left-handed isoform of Watson and Crick’s B-DNA, is rarely formed without the help of high salt concentrations or negative supercoiling. However, Z-DNA-binding proteins can efficiently convert specific sequences of the B conformation into the Z conformation in relaxed DNA under physiological salt conditions. As in the case of many other specific interactions coupled with structural rearrangements in biology, it has been an intriguing question whether the proteins actively induce Z-DNAs or passively trap transiently preformed Z-DNAs. In this study, we used single-molecule fluorescence assays to observe intrinsic B-to-Z transitions, protein association/dissociation events, and accompanying B-to-Z transitions. The results reveal that intrinsic Z-DNAs are dynamically formed and effectively stabilized by Z-DNA-binding proteins through efficient trapping of the Z conformation rather than being actively induced by them. Our study provides, for the first time, detailed pictures of the intrinsic B-to-Z transition dynamics and protein-induced B-to-Z conversion mechanism at the single-molecule level.

Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites

UNLABELLED We present Cas-Designer, a user-friendly program to aid researchers in choosing appropriate target sites in a gene of interest for type II CRISPR/Cas-derived RNA-guided endonucleases, which are now widely used for biomedical research and biotechnology. Cas-Designer rapidly provides the list of all possible guide RNA sequences in a given input DNA sequence and their potential off-target sites including bulge-type sites in a genome of choice. In addition, the program assigns an out-of-frame score to each target site to help users choose appropriate sites for gene knockout. Cas-Designer shows the results in an interactive table and provides user-friendly filter functions. AVAILABILITY AND IMPLEMENTATION Free access at http://rgenome.net/cas-designer/.

Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease

The type II CRISPR-associated protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex. For the Cas9:gRNA complex, we identify sub-conformations of the RNA–DNA heteroduplex during R-loop expansion. Our single-molecule study indicates that the kinetics of the sub-conformations is controlled by the complementarity between crRNA and target DNA. We conclude that both tracrRNA and crRNA regulate the conformations and kinetics of the Cas9 complex, which are crucial in the DNA cleavage activity of the CRISPR-Cas9 system.

Distinct Z-DNA binding mode of a PKR-like protein kinase containing a Z-DNA binding domain (PKZ)

Double-stranded ribonucleic acid-activated protein kinase (PKR) downregulates translation as a defense mechanism against viral infection. In fish species, PKZ, a PKR-like protein kinase containing left-handed deoxyribonucleic acid (Z-DNA) binding domains, performs a similar role in the antiviral response. To understand the role of PKZ in Z-DNA recognition and innate immune response, we performed structural and functional studies of the Z-DNA binding domain (Zα) of PKZ from Carassius auratus (caZαPKZ). The 1.7-Å resolution crystal structure of caZαPKZ:Z-DNA revealed that caZαPKZ shares the overall fold with other Zα, but has discrete structural features that differentiate its DNA binding mode from others. Functional analyses of caZαPKZ and its mutants revealed that caZαPKZ mediates the fastest B-to-Z transition of DNA among Zα, and the minimal interaction for Z-DNA recognition is mediated by three backbone phosphates and six residues of caZαPKZ. Structure-based mutagenesis and B-to-Z transition assays confirmed that Lys56 located in the β-wing contributes to its fast B-to-Z transition kinetics. Investigation of the DNA binding kinetics of caZαPKZ further revealed that the B-to-Z transition rate is positively correlated with the association rate constant. Taking these results together, we conclude that the positive charge in the β-wing largely affects fast B-to-Z transition activity by enhancing the DNA binding rate.

Pharmacokinetics of oltipraz in diabetic rats with liver cirrhosis

Background and purpose:  The incidence of diabetes mellitus is increased in patients with liver cirrhosis. Oltipraz is currently in trials to treat patients with liver fibrosis and cirrhosis induced by chronic hepatitis types B and C and is primarily metabolized via hepatic cytochrome P450 isozymes CYP1A1/2, 2B1/2, 2C11, 2D1 and 3A1/2 in rats. We have studied the influence of diabetes mellitus on pharmacokinetics of oltipraz and on expression of hepatic, CYP1A, 2B1/2, 2C11, 2D and 3A in rats with experimental liver cirrhosis.