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"Scott, David A."
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Implications of human genetic variation in CRISPR-based therapeutic genome editing
Analysis of the ExAC and 1000 Genomes data sets estimates the impact of inter-individual variation on the efficacy and safety of therapies based on CRISPR endonucleases.
CRISPR–Cas genome-editing methods hold immense potential as therapeutic tools to fix disease-causing mutations at the level of DNA. In contrast to typical drug development strategies aimed at targets that are highly conserved among individual patients, treatment at the genomic level must contend with substantial inter-individual natural genetic variation. Here we analyze the recently released ExAC and 1000 Genomes data sets to determine how human genetic variation impacts target choice for Cas endonucleases in the context of therapeutic genome editing. We find that this genetic variation confounds the target sites of certain Cas endonucleases more than others, and we provide a compendium of guide RNAs predicted to have high efficacy in diverse patient populations. For further analysis, we focus on 12 therapeutically relevant genes and consider how genetic variation affects off-target candidates for these loci. Our analysis suggests that, in large populations of individuals, most candidate off-target sites will be rare, underscoring the need for prescreening of patients through whole-genome sequencing to ensure safety. This information can be integrated with empirical methods for guide RNA selection into a framework for designing CRISPR-based therapeutics that maximizes efficacy and safety across patient populations.
Journal Article
Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells
The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.
Journal Article
The soccer surprise
\"A famous soccer star is coming to town, but can practice with a pro help the MVP kids score big at their next game?\"-- Provided by publisher.
Book
DNA targeting specificity of RNA-guided Cas9 nucleases
Analyses of the determinants of the specificity of Cas9 nuclease provide rules for selecting optimal target sites.
The
Streptococcus pyogenes
Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing
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. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
Journal Article
The gold medal mess
\"Five friends are ready for their school's Olympics field day. But not everyone wants to play fair: someone is trying to ruin the events. Can the kids in the Most Valuable Player club solve the mystery, save the Olympics, and take home the gold?\"-- Provided by publisher.
Book
Optical control of mammalian endogenous transcription and epigenetic states
Here the customizable TALE DNA-binding domain was integrated with the light-sensitive cryptochrome 2 protein and its interacting partner (CIB1) from
Arabidopsis thaliana
, thereby creating an optogenetic two-hybrid system called light-inducible transcriptional effectors (LITEs); the LITE system establishes a novel mode of optogenetic control of endogenous transcription and epigenetic states.
LITE hybrids shine in optogenetics
Feng Zhang and colleagues integrated the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from
Arabidopsis thaliana
, which they call LITEs (light-inducible transcriptional effectors), thereby creating an optogenetic two-hybrid system. LITEs do not require additional cofactors, are readily customized to target many loci and can be quickly and reversibly activated. They can also be packaged into viral vectors and targeted to specific cell populations. The authors applied this system in primary mouse neurons and in the brains of awake mice to modulate endogenous gene expression and to target epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes.
The dynamic nature of gene expression enables cellular programming, homeostasis and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high-precision spatiotemporal control of many cellular functions
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. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here we describe the development of light-inducible transcriptional effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain
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with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from
Arabidopsis thaliana
. LITEs do not require additional exogenous chemical cofactors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility
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. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of freely behaving mice
in vivo
to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.
Journal Article
CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus
Chronic hepatitis B virus (HBV) infection is prevalent, deadly and seldom cured due to the persistence of viral episomal DNA (cccDNA) in infected cells. Newly developed genome engineering tools may offer the ability to directly cleave viral DNA, thereby promoting viral clearance. Here, we show that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in robust suppression of viral gene expression and replication. Upon sustained expression of Cas9 and appropriately chosen guide RNAs, we demonstrate cleavage of cccDNA by Cas9 and a dramatic reduction in both cccDNA and other parameters of viral gene expression and replication. Thus, we show that directly targeting viral episomal DNA is a novel therapeutic approach to control the virus and possibly cure patients.
Journal Article