Biology subjects

Feng Zhang

Publications and source records attributed to Feng Zhang.

7 recordsLinked to original sources

bioRxiv2016

Multiplex gene editing by CRISPR-Cpf1 through autonomous processing of a single crRNA array

Microbial CRISPR-Cas defense systems have been adapted as a platform for genome editing applications built around the RNA-guided effector nucleases, such as Cas9. We recently reported the characterization of Cpf1, the effector nuclease of a novel type V-A CRISPR system, and demonstrated that it can be adapted for genome editing in mammalian cells (Zetsche et al., 2015). Unlike Cas9, which utilizes a trans-activating crRNA (tracrRNA) as well as the endogenous RNaseIII for maturation of its dual crRNA:tracrRNA guides (Deltcheva et al., 2011), guide processing of the Cpf1 system proceeds in the absence of tracrRNA or other Cas (CRISPR associated) genes (Zetsche et al., 2015) (Figure 1a), suggesting that Cpf1 is sufficient for pre-crRNA maturation. This has important implications for genome editing, as it would provide a simple route to multiplex targeting. Here, we show for two Cpf1 orthologs that no other factors are required for array processing and demonstrate multiplex gene editing in mammalian cells as well as in the mouse brain by using a designed single CRISPR array.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC=\"FIGDIR/small/049122_fig1.gif\" ALT=\"Figure 1\">\nView larger version (35K):\norg.highwire.dtl.DTLVardef@1f0c15corg.highwire.dtl.DTLVardef@126a6fdorg.highwire.dtl.DTLVardef@9d6a2eorg.highwire.dtl.DTLVardef@a61f75_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1C_FLOATNO Cpf1 mediates processing of pre-crRNA. (a) Schematic of pre-crRNA processing for Cas9 and Cpf1. Cleavage sites indicated with red triangle. (b) In vitro processing of FnCpf1 pre-crRNA transcript (80 nM) with purified AsCpf1 or LbCpf1 protein ([~]320 nM). In the presence of Cpf1 nuclease the pre-crRNA was cleaved in a distinct pattern, indicating cleavage at similar sequence motifs. RNA molecules without Cpf1 DR features where not cleaved by Cpf1 (control RNA). (c) RNAseq analysis of FnCpf1 pre-crRNA cleavage products, as shown in (b). A high fraction of sequence reads smaller than 65nt are cleavage products of spacers flanked by DR sequences.\n\nC_FIG

Molecular Biology↗

bioRxiv2016

Genome Editing With Targeted Deaminases

Precise genetic modifications are essential for biomedical research and gene therapy. Yet, traditional homology-directed genome editing is limited by the requirements for DNA cleavage, donor DNA template and the endogenous DNA break-repair machinery. Here we present programmable cytidine deaminases that enable site-specific cytidine to thymidine (C-to-T) genomic edits without the need for DNA cleavage. Our targeted deaminases are efficient and specific in Escherichia coli, converting a genomic C-to-T with 13% efficiency and 95% accuracy. Edited cells do not harbor unintended genomic abnormalities. These novel enzymes also function in human cells, leading to a site-specific C-to-T transition in 2.5% of cells with reduced toxicity compared with zinc-finger nucleases. Targeted deaminases therefore represent a platform for safer and effective genome editing in prokaryotes and eukaryotes, especially in systems where DSBs are toxic, such as human stem cells and repetitive elements targeting.

Bioengineering↗

bioRxiv2016

Protocol: Genome-scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial immune system CRISPR (clustered regularly interspaced short palindromic repeats) has been adapted for genome-scale screening by combining Cas9 with guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom-or ready-made guide RNA libraries are constructed and packaged into lentivirus for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified from the screen, we further describe strategies for confirming the screening phenotype as well as genetic perturbation through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 6-10 weeks followed by 3-4 weeks of validation.

Molecular Biology↗

bioRxiv2016

C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector

The CRISPR-Cas adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the Class 2 type VI-A CRISPR-Caseffector C2c2 and demonstrate its RNA-guided RNase function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage.In vitro biochemical analysis show that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved HEPN domains, mutations in which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.

Molecular Biology↗

bioRxiv2016

High-resolution interrogation of functional elements in the noncoding genome

The noncoding genome plays a major role in gene regulation and disease yet we lack tools for rapid identification and manipulation of noncoding elements. Here, we develop a large-scale CRISPR screen employing ~18,000 sgRNAs targeting >700 kb of noncoding sequence in an unbiased manner surrounding three genes (NF1, NF2, and CUL3) involved in resistance to the BRAF inhibitor vemurafenib in the BRAF-mutant melanoma cell line A375. We identify specific noncoding locations near genes that modulate drug resistance when mutated. These sites have predictive hallmarks of noncoding function, such as physical interaction with gene promoters, evolutionary conservation and tissue-specific chromatin accessibility. At a subset of identified elements at the CUL3 locus, we show that engineered mutations lead to a loss of gene expression associated with changes in transcription factor occupancy and in long-range and local epigenetic environments, implicating these sites in gene regulation and chemotherapeutic resistance. This demonstration of an unbiased mutagenesis screen across large noncoding regions expands the potential of pooled CRISPR screens for fundamental genomic discovery and for elucidating biologically relevant mechanisms of gene regulation.

Molecular Biology↗

bioRxiv2016

Div-Seq: A single nucleus RNA-Seq method reveals dynamics of rare adult newborn neurons in the CNS

Transcriptomes of individual neurons provide rich information about cell types and dynamic states. However, it is difficult to capture rare dynamic processes, such as adult neurogenesis, because isolation from dense adult tissue is challenging, and markers for each phase are limited. Here, we developed Div-Seq, which combines Nuc-Seq, a scalable single nucleus RNA-Seq method, with EdU-mediated labeling of proliferating cells. We first show that Nuc-Seq can sensitively identify closely related cell types within the adult hippocampus. We apply Div-Seq to track transcriptional dynamics of newborn neurons in an adult neurogenic region in the hippocampus. Finally, we find rare adult newborn GABAergic neurons in the spinal cord, a non-canonical neurogenic region. Taken together, Nuc-Seq and Div-Seq open the way for unbiased analysis of any complex tissue.

Neuroscience↗

bioRxiv2014

Improved vectors and genome-wide libraries for CRISPR screening

Genome-wide, targeted loss-of-function pooled screens using the CRISPR (clustered regularly interspaced short palindrome repeats)-associated nuclease Cas9 in human and mouse cells provide an alternative screening system to RNA interference (RNAi) and have been used to reveal new mechanisms in diverse biological models1-4. Previously, we used a Genome-scale CRISPR Knock-Out (GeCKO) library to identify loss-of-function mutations conferring vemurafenib resistance in a melanoma model1. However, initial lentiviral delivery systems for CRISPR screening had low viral titer or required a cell line already expressing Cas9, limiting the range of biological systems amenable to screening.

Genomics↗