, Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest

J. A. Gilbert, M. J. Blaser, J. G. Caporaso, J. K. Jansson, S. V. Lynch et al., Current understanding of the human microbiome, Nat Med, vol.24, pp.392-400, 2018.

R. Barrangou, C. Fremaux, H. Deveau, M. Richards, P. Boyaval et al., CRISPR provides acquired resistance against viruses in prokaryotes, Science, vol.80, pp.1709-1712, 2007.

K. S. Makarova, Y. I. Wolf, J. Iranzo, S. A. Shmakov, O. S. Alkhnbashi et al., Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants, Nat Rev Microbiol, vol.18, pp.67-83, 2019.

M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Science, vol.337, pp.816-821, 2012.

W. Jiang, D. Bikard, D. Cox, F. Zhang, and L. A. Marraffini, RNA-guided editing of bacterial genomes using CRISPR-Cas systems, Nat Biotechnol, vol.31, pp.233-239, 2013.

L. A. Gilbert, M. H. Larson, L. Morsut, Z. Liu, G. A. Brar et al., CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes, Cell, vol.154, pp.442-451, 2013.

D. Bikard, W. Jiang, P. Samai, A. Hochschild, F. Zhang et al., Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system, Nucleic Acids Res, vol.41, pp.7429-7437, 2013.

L. S. Qi, M. H. Larson, L. A. Gilbert, J. A. Doudna, J. S. Weissman et al., Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression, Cell, vol.152, pp.1173-1183, 2013.

L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto et al., Multiplex genome engineering using CRISPR/Cas systems, Science, p.339

P. Mali, L. Yang, K. M. Esvelt, J. Aach, M. Guell et al., RNA-guided human genome engineering via Cas9, Science, vol.339, pp.823-826

J. Bradford and D. Perrin, A benchmark of computational CRISPR-Cas9 guide design methods, PLoS Comput Biol, vol.15, p.1007274, 2019.

J. Bradford and D. Perrin, Improving CRISPR guide design with consensus approaches, BMC Genomics, vol.20, p.931, 2019.

O. Shalem, N. E. Sanjana, E. Hartenian, X. Shi, D. A. Scott et al., Genome-scale CRISPR-Cas9 knockout screening in human cells, Science, vol.343, pp.84-87, 2014.

T. Wang, J. J. Wei, D. M. Sabatini, and E. S. Lander, Genetic screens in human cells using the CRISPR-Cas9 system, Science, vol.343, pp.80-84, 2014.

H. Koike-yusa, Y. Li, E. Tan, M. Velasco-herrera, and K. Yusa, Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library, Nat Biotechnol, vol.32, pp.267-273, 2014.

A. R. Pacheco, J. E. Lazarus, B. Sit, S. Schmieder, W. I. Lencer et al., CRISPR screen reveals that EHEC's T3SS and Shiga toxin rely on shared host factors for infection, vol.9, pp.1003-1021, 2018.

, authors identify human genes required for the toxicity of enterohemorragic E. coli in intestinal epithelial cells. Interestingly, a common route for the toxicity of the shiga-toxin and the type-3 secretion system is unveiled

J. S. Park, J. D. Helble, J. E. Lazarus, G. Yang, C. J. Blondel et al., A FACS-based genome-wide CRISPR screen reveals a requirement for COPI in Chlamydia trachomatis invasion, vol.11, pp.71-84, 2019.

L. A. Gilbert, M. A. Horlbeck, B. Adamson, J. E. Villalta, Y. Chen et al., Genome-scale CRISPR-mediated control of gene repression and activation, Cell, vol.159, pp.647-661, 2014.

Y. Zhou, S. Zhu, C. Cai, P. Yuan, C. Li et al., Highthroughput screening of a CRISPR/Cas9 library for functional genomics in human cells, Nature, vol.509, pp.487-491, 2014.

L. Tao, J. Zhang, P. Meraner, A. Tovaglieri, X. Wu et al., Frizzled proteins are colonic epithelial receptors for C. difficile toxin B, Nature, vol.538, pp.350-355, 2016.

C. J. Blondel, J. S. Park, T. P. Hubbard, A. R. Pacheco, C. J. Kuehl et al., CRISPR/Cas9 screens reveal requirements for host cell sulfation and fucosylation in bacterial Type III secretion system-mediated cytotoxicity, Cell Host Microbe, vol.20, pp.226-237, 2016.

J. M. Peters, A. Colavin, H. Shi, T. L. Czarny, M. H. Larson et al., A comprehensive, CRISPR-based functional analysis of essential genes in bacteria, Cell, vol.165, pp.1493-1506, 2016.

X. Liu, C. Gallay, M. Kjos, A. Domenech, J. Slager et al., High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae, vol.13, p.931, 2017.

T. J. De-wet, K. R. Winkler, M. M. Mhlanga, V. Mizrahi, and D. F. Warner, Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes, 2020.

F. Caro, N. M. Place, and J. J. Mekalanos, Analysis of lipoprotein transport depletion in Vibrio cholerae using CRISPRi, Proc Natl Acad Sci, vol.116, pp.17013-17022, 2019.

T. Wang, J. Guo, Y. Liu, Z. Xue, C. Zhang et al., Genome-wide screening identifies promiscuous phosphatases impairing terpenoid biosynthesis in Escherichia coli, Appl Microbiol Biotechnol, vol.102, pp.9771-9780, 2018.

H. S. Rishi, E. Toro, H. Liu, X. Wang, L. S. Qi et al., Systematic genome-wide querying of coding and non-coding functional elements in E. coli using CRISPRi, 2020.

F. Rousset, J. C. Caballero, F. Piastra-facon, J. Ferná-ndez-rodríguez, O. Clermont et al., The impact of genetic diversity on gene essentiality within the E. coli species, 2020.

L. Yao, K. Shabestary, S. M. Bjö-rk, J. Asplund-samuelsson, H. N. Joensson et al., Pooled CRISPRi screening of the cyanobacterium Synechocystis sp. PCC 6803 for enhanced industrial phenotypes, Nat Commun, vol.2020, p.1666

, A nice application of CRISPRi screens in a biotechnological context. In particular, the authors combine droplet-based cell sorting with a chemical assay to identify knockdowns that produce higher titers of L-lactate

T. J. De-wet, I. Gobe, M. M. Mhlanga, and D. F. Warner, CRISPRi-Seq for the identification and characterisation of essential mycobacterial genes and transcriptional units, 2018.

H. H. Lee, N. Ostrov, B. G. Wong, M. A. Gold, A. S. Khalil et al., Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi, vol.4, pp.1105-1113, 2019.

, This study uses CRISPRi to identify the essential genes of V. natriegens, a species of biotechnological interest for which Tn-seq was unsuccessfull

D. Beuter, J. V. Gomes-filho, L. Randau, F. Díaz-pascual, K. Drescher et al., Selective enrichment of slow-growing bacteria in a metabolism-wide CRISPRi library with a TIMER protein, ACS Synth Biol, vol.7, pp.2775-2782, 2018.

S. Li, C. B. Jendresen, J. Landberg, L. E. Pedersen, N. Sonnenschein et al., Genome-wide CRISPRi-based identification of targets for decoupling growth from production, ACS Synth Biol, vol.2020, pp.1030-1040

D. Camsund, M. J. Lawson, J. Larsson, D. Jones, S. Zikrin et al., accessing certain phenotypes is challenging with pooled CRISPRi screens, the authors provide an ingenious solution by combining timelapse microscopy of a pooled library with in situ genotyping by sequential FISH, Nat Methods, vol.17, pp.86-92, 2019.

W. Jiang, P. Oikonomou, and S. Tavazoie, Comprehensive genomewide perturbations via CRISPR adaptation reveal complex genetics of antibiotic sensitivity, Cell, vol.2020, p.0

J. M. Peters, B. Koo, R. Patino, G. E. Heussler, C. C. Hearne et al., Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi, Nat Microbiol, vol.4, pp.244-250, 2019.

J. Qu, N. K. Prasad, M. A. Yu, S. Chen, A. Lyden et al., Modulating pathogenesis with mobile-CRISPRi, J Bacteriol, 0201.

A. K. Cain, L. Barquist, A. L. Goodman, I. T. Paulsen, J. Parkhill et al., A decade of advances in transposon-insertion sequencing, Nat Rev Genet, 2020.

X. Liu, J. M. Kimmey, V. Bakker, . De, V. Nizet et al., Exploration of bacterial bottlenecks and Streptococcus pneumoniae pathogenesis by CRISPRi-seq, 2020.

, This preprint describes the first CRISPRi screen employed in vivo in bacteria, investigating the population bottlenecks during pneumococcal infection in mice and identifying pneumococcal genes required for pathogenicity

B. F. Cress, Ö. Toparlak, S. Guleria, M. Lebovich, J. T. Stieglitz et al., CRISPathBrick: modular combinatorial assembly of Type II-A CRISPR arrays for dCas9-mediated multiplex transcriptional repression in E. coli, ACS Synth Biol, vol.4, pp.987-1000, 2015.

M. L. Maeder, S. J. Linder, V. M. Cascio, Y. Fu, Q. H. Ho et al., CRISPR RNA-guided activation of endogenous human genes, Nat Methods, vol.10, pp.977-979, 2013.

C. Dong, J. Fontana, A. Patel, J. M. Carothers, and J. G. Zalatan, Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria, Nat Commun, vol.9, p.2489, 2018.

Y. Liu, X. Wan, and B. Wang, Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria, Nat Commun, vol.10, p.3693, 2019.

J. Fontana, C. Dong, C. Kiattisewee, V. P. Chavali, B. I. Tickman et al., Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements, Nat Commun, vol.11, pp.1-11, 2020.

H. Ho, J. Fang, J. Cheung, and H. H. Wang, Programmable and portable CRISPR-Cas transcriptional activation in bacteria, 2020.

A. C. Komor, Y. B. Kim, M. S. Packer, J. A. Zuris, and D. R. Liu, Programmable editing of a target base in genomic DNA without doublestranded DNA cleavage, Nature, vol.533, pp.420-424, 2016.

N. M. Gaudelli, A. C. Komor, H. A. Rees, M. S. Packer, A. H. Badran et al., Programmable base editing of T to G C in genomic DNA without DNA cleavage, Nature, vol.551, pp.464-471, 2017.

T. Gu, S. Zhao, Y. Pi, W. Chen, C. Chen et al., Highly efficient base editing in: Staphylococcus aureus using an engineered CRISPR RNA-guided cytidine deaminase, Chem Sci, vol.9, pp.3248-3253, 2018.

Y. Zhang, H. Zhang, Z. Wang, Z. Wu, Y. Wang et al., Programmable adenine deamination in bacteria using a Cas9-adenine-deaminase fusion, Chem Sci, vol.11, pp.1657-1664, 2020.

Y. Wang, S. Wang, W. Chen, L. Song, Y. Zhang et al., CRISPRCas9 and CRISPR-assisted cytidine deaminase enable precise and efficient genome editing in Klebsiella pneumoniae, Appl Environ Microbiol, p.84, 2018.

Y. Wang, Z. Wang, Y. Chen, X. Hua, Y. Yu et al., A highly efficient CRISPR-Cas9-based genome engineering platform in Acinetobacter baumannii to understand the H 2 O 2 -sensing mechanism of OxyR, Cell Chem Biol, vol.26, pp.1732-1742, 2019.

W. Chen, Y. Zhang, Y. Zhang, Y. Pi, T. Gu et al., CRISPR/Cas9-based genome editing in Pseudomonas aeruginosa and cytidine deaminase-mediated base editing in Pseudomonas species, vol.6, pp.222-231, 2018.

Y. Tong, C. M. Whitford, H. L. Robertsen, K. Blin, T. S. Jørgensen et al., Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST

, Proc Natl Acad Sci, vol.116, pp.20366-20375, 2019.

S. Banno, K. Nishida, T. Arazoe, H. Mitsunobu, and A. Kondo, Deaminase-mediated multiplex genome editing in Escherichia coli, Nat Microbiol, vol.3, pp.423-429, 2018.

E. Sharon, S. Chen, N. M. Khosla, J. D. Smith, J. K. Pritchard et al., Functional genetic variants revealed by massively parallel precise genome editing, Cell, vol.175, pp.544-557, 2018.

H. Lim, S. Jun, M. Park, J. Lim, J. Jeong et al., Multiplex generation, tracking, and functional screening of substitution mutants using a crispr/retron system, ACS Synth Biol, vol.2020, pp.1003-1009

A. V. Anzalone, P. B. Randolph, J. R. Davis, A. A. Sousa, L. W. Koblan et al., Search-and-replace genome editing without double-strand breaks or donor DNA, Nature, vol.576, pp.149-157, 2019.

J. Strecker, A. Ladha, Z. Gardner, J. L. Schmid-burgk, K. S. Makarova et al., RNA-guided DNA insertion with CRISPRassociated transposases, Science, vol.364, pp.48-53

S. E. Klompe, P. Vo, T. S. Halpin-healy, and S. H. Sternberg, Transposon-encoded CRISPR-Cas systems direct RNAguided DNA integration, Nature, vol.571, pp.219-225, 2019.