CRISPR/Cas is a defense system that prokaryotes deploy to battle invading viruses. This system has been engineered to serve labs around the world as tools to edit the genome.
The Royal Swedish Academy of Sciences has just awarded the Nobel Prize in Chemistry 2020 to Emmanuelle Charpentier of the Max Planck Unit for the Science of Pathogens in Berlin, Germany and to Jennifer Doudna of the University of California, Berkeley "for the development of a method for genome editing." This is about harnessing CRISPR/Cas9 to alter the genomes of animals, plants and microbes.
We congratulate the winners, are happy for them and for all scientists who work in this burgeoning field. Here are some of the papers, research highlights and journalistic pieces we have published in this area and we look forward to many more. Note: I compiled this page with the help of my awesome colleague Lei Tang.
The CRISPR-Cas9 system is best known for its ability to knock out or replace specific genes, via targeted cleavage of the genome. But scientists are developing many more applications, typically by using an inactive Cas9 to target other enzymes to specific genomic sites. From transcriptional regulation to base editing, these developments are extending the range of biological questions that can be probed with CRISPR/Cas9.
See a poster here. The poster shows the steps through which CRISPR-Cas9 can be used to knock out or replace specific genes. And it also presents a bundle of applications such as transcriptional regulation, epigenome editing and base editing. Made in collaboration with Stanford University researchers Josh Tycko, Gaelen T Hess, Edwin E Jeng, Michael Dubreuil and Michael C Bassik
Watch a video here. Scientific advisors: Nobel Laureate Jennifer Doudna from the University of California Berkeley and Megan Hochstrasser from the Innovative Genomics Institute. Scientific Lead: Nicole Rusk, Nature Methods. (Produced with support from Horizon).
The above image and the image here are from this video.
Kevin M Esvelt, Prashant Mali, Jonathan L Braff, Mark Moosburner, Stephanie J Yaung and George M Church
…”By experimentally characterizing and demonstrating orthogonality among multiple Cas9 proteins in bacteria and human cells, we have substantially expanded the repertoire of orthogonal RNA-guided DNA-binding elements and constructed a pipeline for characterizing additional examples. “…
2020 – Review
Nicole D. Marino, Rafael Pinilla-Redondo, Bálint Csörgő and Joseph Bondy-Denomy
“The discovery of protein inhibitors of CRISPR-Cas systems, called anti-CRISPR (Acr) proteins, enables the development of more controllable and precise CRISPR-Cas tools. Here we discuss applications of Acr proteins for post-translational control of CRISPR-Cas systems in prokaryotic and mammalian cells, organisms and ecosystems.”
Gigi C. G. Choi , Peng Zhou , Chaya T. L. Yuen, Becky K. C. Chan , Feng Xu , Siyu Bao , Hoi Yee Chu , Dawn Thean , Kaeling Tan, Koon Ho Wong , Zongli Zheng , and Alan S. L. Wong
“Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells.”
2013 - Review
Prashant Mali, Kevin M Esvelt and George M Church
“Looking forward, the versatility and ease of use afforded by Cas9 coupled with its singular ability to bring together RNA, DNA and protein in a fully programmable fashion will form the basis of a powerful toolset for the perturbation, regulation and monitoring of complex biological systems.”
2020 - Research highlight
PAM-less is more Lei Tang
Structure-guided engineering allows researchers access to an expanding number of Cas9 variants that relax the constraint imposed by target site recognition of a protospacer-adjacent motif (PAM).
GUIDE RNA DESIGN AND EVALUATION
Georg Michlits, Julian Jude , Matthias Hinterndorfer, Melanie de Almeid, Gintautas Vainorius, Maria Hubmann, Tobias Neumann, Alexander Schleiffer , Thomas Rainer Burkard , Michaela Fellner, Max Gijsbertsen, Anna Traunbauer , Johannes Zuber and Ulrich Elling
We integrate protein features into a ‘Bioscore’ and fuse it with improved predictors of single-guide RNA activity and indel formation to establish a score that captures all relevant processes in CRISPR–Cas9 mutagenesis. This Vienna Bioactivity CRISPR score (www.vbc-score.org) outperforms previous prediction tools and enables the selection of sgRNAs that effectively produce loss-of-function alleles."
Florian Heigwer, Grainne Kerr and Michael Boutros
“Here we describe E-CRISP, a web application to design gRNA sequences …. It provides flexible output and experiment-oriented design parameters, enabling design of multiple libraries and thereby systematic analysis of the influence of different parameters.”
Samira Kiani, Alejandro Chavez, Marcelle Tuttle, Richard N Hall, Raj Chari, Dmitry Ter-Ovanesyan, Jason Qian, Benjamin W Pruitt, Jacob Beal, Suhani Vora, Joanna Buchthal, Emma J K Kowal, Mohammad R Ebrahimkhani, James J Collins, Ron Weiss and George Church
“We demonstrate that by altering the length of Cas9- associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. “
Raj Chari, Prashant Mali, Mark Moosburner and George M Church
“We developed an in vivo library-on-library methodology to simultaneously assess single guide RNA (sgRNA) activity across ~1,400 genomic loci. …Our results and software (http://crispr.med. harvard.edu/sgRNAScorer) enable improved design of reagents, shed light on mechanisms of genome targeting, and provide a generalizable framework to study nucleic acid–nucleic acid interactions and biochemistry in high throughput.”
Miguel A Moreno-Mateos, Charles E Vejnar, Jean-Denis Beaudoin, Juan P Fernandez, Emily K Mis, Mustafa K Khokha and Antonio J Giraldez
These results identify determinants that influence Cas9 activity and provide a framework for the design of highly efficient sgRNAs for genome targeting in vivo."
IN VIVO EDITING
Daniel J Dickinson, Jordan D Ward, David J Reiner and Bob Goldstein
Ari E Friedland, Yonatan B Tzur, Kevin M Esvelt, Monica P Colaiácovo, George M Church and John A Calarco
Wei Leong Chew, Mohammadsharif Tabebordbar, Jason K W Cheng, Prashant Mali, Elizabeth Y Wu, Alex H M Ng, Kexian Zhu, Amy J Wagers an George M Church
Bin Shen, Wensheng Zhang, Jun Zhang, Jiankui Zhou, Jianying Wang, Li Chen, Lu Wang, Alex Hodgkins, Vivek Iyer, Xingxu Huang & William C Skarnes
Co-microinjection of mouse embryos with Cas9 mRNA and single guide RNAs induces on-target and off-target mutations that are transmissible to offspring. However, Cas9 nickase can be used to efficiently mutate genes without detectable damage at known off-target sites. "
Jeffrey C Wagner, Randall J Platt, Stephen J Goldfless, Feng Zhang & Jacquin C Niles
Hui K Kim, Myungjae Song, Jinu Lee, A Vipin Menon, Soobin Jung, Young-Mook Kang, Jae W Choi, Euijeon Woo, Hyun C Koh, Jin-Wu Nam & Hyongbum Kim
TOOLS TO EVALUATE EDITING OUTCOMES
Daesik Kim, Sangsu Bae, Jeongbin Park, Eunji Kim, Seokjoong Kim, Hye Ryeong Yu, Jinha Hwang, Jong-Il Kim & Jin-Soo Kim
“Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9."
Peter Cameron, Chris K Fuller, Paul D Donohoue, Brittnee N Jones, Matthew S Thompson, Matthew M Carter, Scott Gradia, Bastien Vidal, Elizabeth Garner1, Euan M Slorach, Elaine Lau, Lynda M Banh, Alexandra M Lied, Leslie S Edwards, Alexander H Settle, Daniel Capurso, Victor Llaca, Stéphane Deschamps, Mark Cigan, Joshua K Young & Andrew P May
Arne H. Smits, Frederik Ziebell, Gerard Joberty , Nico Zinn , William F. Mueller, Sandra Clauder-Münster, Dirk Eberhard , Maria Fälth Savitski , Paola Grandi , Petra Jakob , Anne-Marie Michon , Hanice Sun , Karen Tessmer, Tilmann Bürckstümmer, Marcus Bantscheff , Lars M. Steinmetz, Gerard Drewes and Wolfgang Huber
Our results imply that systematic characterization of residual protein expression or function in CRISPR–Cas9-generated KO lines is necessary for phenotype interpretation."
Shengdar Q Tsai, Nhu T Nguyen, Jose Malagon-Lopez, Ved V Topkar, Martin J Aryee & J Keith Joung
“Here we describe circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR–Cas9 genome-wide off-target mutations.”
BEYOND EDITING – Gene Activation
Pablo Perez-Pinera, D Dewran Kocak, Christopher M Vockley, Andrew F Adler, Ami M Kabadi, Lauren R Polstein, Pratiksha I Thakore, Katherine A Glass, David G Ousterout, Kam W Leong, Farshid Guilak, Gregory E Crawford, Timothy E Reddy & Charles A Gersbach
In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation."
Alejandro Chavez, Marcelle Tuttle, Benjamin W Pruitt, Ben Ewen-Campen, Raj Chari, Dmitry Ter-Ovanesyan, Sabina J Haque, Ryan J Cecchi, Emma J K Kowal, Joanna Buchthal, Benjamin E Housden, Norbert Perrimon, James J Collins and George Church
Our results demonstrate that, across a range of target genes and cellular environments, the VPR, SAM, and Suntag systems are consistently superior to the previous VP64 standard. In addition, while SAM exhibits more potent activation in some contexts, VPR, SAM, and Suntag generally fall within an order of magnitude of each other with regard to fold increase in gene expression. It is additionally comforting that each activator was similarly specific to a given target."
BEYOND EDITING - Transcriptional programming
Alejandro Chavez, Jonathan Scheiman, Suhani Vora, Benjamin W Pruitt, Marcelle Tuttle, Eswar P R Iyer, Shuailiang Lin, Samira Kiani, Christopher D Guzman, Daniel J Wiegand, Dmitry Ter-Ovanesyan, Jonathan L Braff, Noah Davidsohn, Benjamin E Housden, Norbert Perrimon, Ron Weiss, John Aach, James J Collins and George M Church
BEYOND EDITING - Transcriptional activation
Wenkai Yi, Jingyu Li , Xiaoxuan Zhu, Xi Wang , Ligang Fan, Wenju Sun, Linbu Liao , Jilin Zhang , Xiaoyu Li, Jing Ye, Fulin Chen , Jussi Taipale, Kui Ming Chan , Liang Zhang and Jian Yan
BEYOND EDITING – Epigenetic editing
Pratiksha I Thakore, Anthony M D’Ippolito, Lingyun Song, Alexias Safi, Nishkala K Shivakumar, Ami M Kabadi, Timothy E Reddy, Gregory E Crawford and Charles A Gersbach
These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome."
2016 - Review
Pratiksha I Thakore, Joshua B Black, Isaac B Hilton and Charles A Gersbach
“The unique properties of the CRISPR-Cas9 system have created new opportunities for high-throughput genetic screens and multiplexing targets to manipulate complex gene expression patterns. This Review summarizes recent technological developments in this area and their application to biomedical challenges. We also discuss remaining limitations and necessary future directions for this field.”
BEYOND EDITING – Screening
Paul Datlinger, André F Rendeiro, Christian Schmidl, Thomas Krausgruber, Peter Traxler, Johanna Klughammer, Linda C Schuster, Amelie Kuchler, Donat Alpar and Christoph Bock
Here we combine pooled CRISPR screening with single-cell RNA sequencing into a broadly applicable workflow, directly linking guide RNA expression to transcriptome responses in thousands of individual cells. Our method for CRISPR droplet sequencing (CROP-seq) enables pooled CRISPR screens with single-cell transcriptome resolution, which will facilitate high-throughput functional dissection of complex regulatory mechanisms and heterogeneous cell populations."
Arish N Shah, Crystal F Davey, Alex C Whitebirch, Adam C Miller and Cecilia B Moens
“We performed a proof-of concept screen in which we used intersecting, multiplexed pool injections to examine 48 loci and identified two new genes involved in electrical-synapse formation. By deep sequencing target loci, we found that 90% of the genes were effectively screened. We conclude that CRISPR can be used as a powerful reverse genetic screening strategy in vivo in a vertebrate system.”
Eleni P. Mimitou, Anthony Cheng, Antonino Montalbano, Stephanie Hao, Marlon Stoeckius , Mateusz Legut, Timothy Roush, Alberto Herrera , Efthymia Papalexi, Zhengqing Ouyang, Rahul Satija , Neville E. Sanjana, Sergei B. Koralov and Peter Smibert
“Multimodal single-cell assays provide high-resolution snapshots of complex cell populations, but are mostly limited to transcriptome plus an additional modality. Here, we describe expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell.”
Neville E Sanjana, Ophir Shalem and Feng Zhang
“Genome-wide, targeted loss-of-function pooled screens using the clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 in human and mouse cells provide an alternative screening system to RNA interference (RNAi)…. We sought to improve both the lentiviral packaging and choice of guide sequences in our original GeCKO library…, where a pooled library of synthesized oligonucleotides was cloned into a lentiviral backbone containing both the Streptococcus pyogenes Cas9 nuclease and the single guide RNA (sgRNA) scaffold. … These changes resulted in an approximately tenfold increase in functional viral titer over that of lentiCRISPRv1 …”
BEYOND EDITING – RNA-protein interactions
Wenkai Yi, Jingyu Li , Xiaoxuan Zhu, Xi Wang , Ligang Fan, Wenju Sun, Linbu Liao , Jilin Zhang , Xiaoyu Li, Jing Ye, Fulin Chen, Jussi Taipale, Kui Ming Chan , Liang Zhang and Jian Yan
BEYOND EDITING – Hypermutation
Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells
Gaelen T Hess, Laure Frésard, Kyuho Han, Cameron H Lee, Amy Li, Karlene A Cimprich, Stephen B Montgomery and Michael C Bassik
“These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.”
Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display
David M Shechner, Ezgi Hacisuleyman, Scott T Younger and John L Rinn
“To investigate and harness these capabilities, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-Display (CRISP-Disp), a targeted localization method that uses Cas9 to deploy large RNA cargos to DNA loci. We demonstrate that functional RNA domains up to at least 4.8 kb long can be inserted in CRISPR guide RNA at multiple points, allowing the construction of Cas9 complexes with protein-binding cassettes, artificial aptamers, pools of random sequences and natural long noncoding RNAs.”
John Paul Shen, Dongxin Zhao, Roman Sasik, Jens Luebeck, Amanda Birmingham, Ana Bojorquez-Gomez, Katherine Licon, Kristin Klepper, Daniel Pekin, Alex N Beckett, Kyle Salinas Sanchez, Alex Thomas, Chih-Chung Kuo, Dan Du, Assen Roguev, Nathan E Lewis, Aaron N Chang, Jason F Kreisberg, Nevan Krogan, Lei Qi, Trey Ideker and Prashant Mali
We developed a systematic approach to map human genetic networks by combinatorial CRISPR–Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction."
Cem Kuscu, Mahmut Parlak, Turan Tufan, Jiekun Yang, Karol Szlachta, Xiaolong Wei, Rashad Mammadov and Mazhar Adli
“Here, we use CRISPR base editors to knock out genes by changing single nucleotides to create stop codons. We show that the CRISPR-STOP method is an efficient and less deleterious alternative to wild-type Cas9 for gene-knockout studies. …CRISPR-STOP-mediated targeted screening demonstrates comparable efficiency to WT Cas9, which indicates the suitability of our approach for genome-wide functional screenings.”
BEYOND EDITING – Research Highlights
Coupling CRISPR to smart hydrogels - Lei Tang
The CRISPR–Cas system has become a powerful tool in genome editing and now expands its footprint into materials science.
Human CRISPR - Nicole Rusk
A humanized version of the RNA-targeting Cas13 can regulate RNA abundance and translation.
Author File - Profiles
Dec 1 2017 Author File - Profile
Improving single-cell data in CRISPR screens, with inspiration from open-air theater performances. Vivien Marx
Our love of hypothesis-driven ideas can mean that people prefer to verify rather than falsify them, says Ulrich Elling, who works at the interface of genomics, developmental and stem cell biology and has been a group leader at the Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA) since 2014. “We do validation experiments, not failed falsification experiments,” he says. “That is why I love unbiased screens.”
Associated paper 16 Oct 2017
Michlits, G et al. CRISPR-UMI: single-cell lineage tracing of pooled CRISPR–Cas9 screens
CRISPR-UMI is a single-cell lineage-tracing methodology for pooled screening to account for cell heterogeneity. A proof-of-principle CRISPR-UMI negative-selection screen provided increased sensitivity and robustness compared with conventional analysis by accounting for underlying cellular and editing-outcome heterogeneity and detection of outlier clones. CRISPR-UMI boosts the predictive power, sensitivity, and information content of pooled CRISPR screens.
27 Feb 2019
RNA-editing tools from a cricket-playing electrical engineer turned bioengineer. Vivien Marx
“I’ve always felt that RNA editing is just a special place to be, because you have this aspect of tunability and reversibility that you don’t have with DNA targeting,” says Prashant Mali. It’s the tunability that makes these tools attractive also for therapeutic applications. “My core passion or at least my dream is that I can take something to the clinic and have an impact there,” he says. “Of course it’s something that is still far away, but I definitely want to give it my best shot.”
Associated paper 8 Feb 2019
Katrekar, D et al. In vivo RNA editing of point mutations via RNA-guided adenosine deaminases
The paper presents in vivo sequence-specific RNA base editing via adenosine deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). To achieve this, we systematically engineered adRNAs to harness ADARs, and comprehensively evaluated the specificity and activity of the toolsets in vitro and in vivo via two mouse models of human disease. We anticipate that this platform will enable tunable and reversible engineering of cellular RNAs for diverse applications.
19 May 2020
A better base editor with fewer off-target changes, from a die-hard Manchester United fan. Vivien Marx
"Yang often gets ideas for the lab through interaction on social media, usually WeChat, but he prefers face-to-face discussion, he says. In the Jaenisch lab, he liked how valued independent thinking and interaction were and has styled his lab in that vein."
Associated paper 18 May 2020
Cytosine base editors (CBEs) offer a powerful tool for correcting point mutations, yet their DNA and RNA off-target activities have caused concerns in biomedical applications. We describe screens of 23 rationally engineered CBE variants, which reveal mutation residues in the predicted DNA-binding site can dramatically decrease the Cas9-independent off-target effects. Furthermore, we obtained a CBE variant—YE1-BE3-FNLS—that retains high on-target editing efficiency while causing extremely low off-target edits and bystander edits.
Nov. 6 2012
Genome-editing tools storm ahead Vivien Marx
The menu of maturing, diversifying methods calls for careful selections in experimental design.
The scientific community is working on ways to comprehensively identify off-target sites and better decipher how these effects come about. “Once we understand them, then we can contemplate how to reduce them,” Keith Joung says. “But we need to figure out what they are first.”
Gene editing: how to stay on-target with CRISPR Vivien Marx
Efficiently cutting a target sequence to effect a desired change in the genome is one gene-editing task. Knowing where else in the genome a tool might have made its mark is quite another.
In some but not all experiments, off-targets can be mission critical. New assessment methods will help, says Charles Gersbach, a biomedical engineer at Duke University. Predicting off-target sites depends on the target sequence to be edited. Of the many techniques to measure or address specificity, none is “perfect,” says Gersbach."
Jan 29 2015
Stem cells: disease models that show and tell Vivien Marx
Combining gene-editing techniques with induced pluripotent stem cells is both powerful and tricky.
Making gene edits in stem cells can be nontrivial, especially when a new section of DNA is inserted via homologous recombination, says Clive Svendsen. CRISPR has relatively low efficiency in these cells, he says. A researcher might look at 100 gene-edited clones, but only 3 will have the correct gene inserted.
Dec 30 2015
Cell biology: delivering tough cargo into cells Vivien Marx
New approaches expand the range and size of materials that can be inserted into a cell.
“The nice thing about electroporation is that one can deliver very large vectors,” says geneticist Ken Scott at the Baylor College of Medicine (BCM). In his lab, transfection with DNA vectors in cell culture is routine. He does not run into load-bearing constraints in those experiments, in which he uses lentivirus as the vector for nucleic acids for high-throughput screening and which he is scaling up to use for in vivo screening in mice. To do this, he applies a dual piggyBac system, originally discovered in baculovirus, in which a mobile genetic element, a transposon, helps with cargo delivery."
Choosing CRISPR-based screens in cancer Vivien Marx
Many possibilities for parsing cancer emerge when labs combine gene editing and screens. And RNAi retains its spot in the menu of options.
False positives are another challenge with 'classic' CRISPR–Cas9-based screens in cancer research. CRISPR is an important knockout technique for work with aneuploid cancer cell lines, says Ji Luo. But when a region in a cancer genome is amplified by, say, 100 copies, CRISPR–Cas9 will cut many or even all of the copies. This, he says, is not off-target but on-target cutting that cells might not survive."
Oct 1 2018
Base editing a CRISPR way Vivien Marx
A way to gene edit without double-stranded DNA breaks is now entering labs.
Base editing has “completely changed the way we build models in the lab,” says Weill Cornell Medicine researcher Lukas Dow. He hopes base editing will help labs build more accurate cancer models to expand on information from large-scale tumor sequencing."
There is no efficiency level below which an editor is useless or above which an editor is useful for all applications, says David Liu. The metric depends on the target, the intended application, and the downstream biology. A low base-editing efficiency might suffice for labs generating a point mutation in a crop, but to achieve therapeutic benefit in a person a high degree of correction in a target organ might be needed, accompanied by a high base-editing efficiency."
21 Jan 2019
Editing the genome of human embryos is ethically fraught. But some projects show how diligent, ethical work can grow the gene-editing field
On a daily basis, Wiles and his team inject CRISPR reagents into mouse zygotes. “Each CRISPR reagent, each guide, behaves very slightly differently,” says Michael Wiles. A designed guide RNA that seemed perfect can end up cutting poorly, yet a guide designed to cut just ten bases adjacent to that first location might do a better job. Micro-injecting embryos takes great skill, and even with people who do this daily, there is some variability, he says. “The micro-injectors are not robots, they are people,” he says. "
To make embryo editing safe for use in mice or humans, says Stanford University researcher Lars Steinmetz, labs need to screen for unwanted on-target mutations and any off-target mutations “very thoroughly,” with deep, whole-genome sequencing, and look for any other potentially harmful side effects before embryos are implanted. “Quality control is of the utmost importance when working on any system that will ultimately make its way to humans,” he says. The Steinmetz lab prefers CRISPR-mediated gene editing over base editing, given the finding by his colleagues and him that base editing can generate more off-target mutations. These mutations “were signatures of cytidine deaminase activity, and were independent of the guide RNA,” he says. Steinmetz’s co-author Hui Yang, a researcher at Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, has preferred base editing, because it avoids double-stranded breaks (DSBs).