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Author

Heon Seok Kim

Bio: Heon Seok Kim is an academic researcher from Stanford University. The author has contributed to research in topics: CRISPR & Cas9. The author has an hindex of 9, co-authored 19 publications receiving 1875 citations. Previous affiliations of Heon Seok Kim include Seoul National University & Chungbuk National University.
Topics: CRISPR, Cas9, Medicine, Gene, Biology

Papers
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Journal ArticleDOI
TL;DR: 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, and paired nickases induced chromosomal deletions in a targeted manner without causing unwanted translocations.
Abstract: 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.

1,332 citations

Journal ArticleDOI
TL;DR: A simple formula and a computer program are developed to predict the deletion patterns at a given nuclease target site that are associated with microhomology of at least two bases, which can be predicted to achieve efficient gene disruption in cell lines and whole organisms.
Abstract: 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

330 citations

Journal ArticleDOI
TL;DR: The Himalayan point mutation in the Tyr gene was introduced by microinjecting ABE mRNA and an extended gRNA into mouse embryos, obtaining Tyr mutant mice with an albino phenotype and the split ABE gene was delivered to muscle cells in a mouse model of Duchenne muscular dystrophy to correct a nonsense mutation.
Abstract: Adenine base editors (ABEs) composed of an engineered adenine deaminase and the Streptococcus pyogenes Cas9 nickase enable adenine-to-guanine (A-to-G) single-nucleotide substitutions in a guide RNA (gRNA)-dependent manner. Here we demonstrate application of this technology in mouse embryos and adult mice. We also show that long gRNAs enable adenine editing at positions one or two bases upstream of the window that is accessible with standard single guide RNAs (sgRNAs). We introduced the Himalayan point mutation in the Tyr gene by microinjecting ABE mRNA and an extended gRNA into mouse embryos, obtaining Tyr mutant mice with an albino phenotype. Furthermore, we delivered the split ABE gene, using trans-splicing adeno-associated viral vectors, to muscle cells in a mouse model of Duchenne muscular dystrophy to correct a nonsense mutation in the Dmd gene, demonstrating the therapeutic potential of base editing in adult animals.

323 citations

Journal ArticleDOI
TL;DR: Adenine base editor–induced cytosine substitutions occur independently of adenosine conversions with an efficiency of up to 11.2% and reduce the number of suitable targeting sites for high-specificity base editing.
Abstract: Adenine base editors comprise an adenosine deaminase, evolved in vitro, and a Cas9 nickase. Here, we show that in addition to converting adenine to guanine, adenine base editors also convert cytosine to guanine or thymine in a narrow editing window (positions 5-7) and in a confined TC*N sequence context. Adenine base editor-induced cytosine substitutions occur independently of adenosine conversions with an efficiency of up to 11.2% and reduce the number of suitable targeting sites for high-specificity base editing.

82 citations

Journal ArticleDOI
TL;DR: A small molecule probe specific for activated macrophages is reported, called CDg16, and its application to visualizing inflammatory atherosclerotic plaques in vivo is demonstrated and used to image atherosclerosis in mice.
Abstract: Activated macrophages have the potential to be ideal targets for imaging inflammation. However, probe selectivity over non-activated macrophages and probe delivery to target tissue have been challenging. Here, we report a small molecule probe specific for activated macrophages, called CDg16, and demonstrate its application to visualizing inflammatory atherosclerotic plaques in vivo. Through a systematic transporter screen using a CRISPR activation library, we identify the orphan transporter Slc18b1/SLC18B1 as the gating target of CDg16.

49 citations


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Journal ArticleDOI
28 Nov 2014-Science
TL;DR: The power of the CRISPR-Cas9 technology to systematically analyze gene functions in mammalian cells, study genomic rearrangements and the progression of cancers or other diseases, and potentially correct genetic mutations responsible for inherited disorders is illustrated.
Abstract: 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.

4,774 citations

Journal ArticleDOI
TL;DR: A modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells, which will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.
Abstract: Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.

2,930 citations

Journal ArticleDOI
TL;DR: Recently devised sgRNA design rules are used to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results, and a metric to predict off-target sites is developed.
Abstract: CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.

2,866 citations

01 Jan 2011
TL;DR: The sheer volume and scope of data posed by this flood of data pose a significant challenge to the development of efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data.
Abstract: Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

2,187 citations

Journal ArticleDOI
28 Jan 2016-Nature
TL;DR: With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type Sp Cas9 for research and therapeutic applications and suggests a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.
Abstract: CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.

2,031 citations