Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis

Liao, Chunyu; Ttofali, Fani; Slotkowski, Rebecca; Denny, Steven R.; Cecil, Taylor D; Leenay, Ryan T.; Keung, Albert J.; Beisel, Chase L.

doi:10.1038/s41467-019-10747-3

Cited by 95 publications

(129 citation statements)

References 58 publications

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“…2b) 21 . This strategy enabled at least four genetic loci to be edited simultaneously in Saccharomyces cerevisiae, Escherichia coli, and cell-free extract 23 . The intrinsic crRNA-processing capabilities of Cas12a have also been leveraged to process a large number of gRNAs from a single RNA transcript, both constitutively and inducibly ( Fig.…”

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confidence: 99%

“…The intrinsic crRNA-processing capabilities of Cas12a have also been leveraged to process a large number of gRNAs from a single RNA transcript, both constitutively and inducibly ( Fig. 2c) [23][24][25] . Cas12a cleaves pre-crRNA via recognition of hairpin structures formed within the spacer repeats, producing mature crRNAs 26 .…”

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confidence: 99%

“…Multiplexed targeting via Cas12a has also been demonstrated in plants, yeast, and bacteria [27][28][29] . In addition, spacers for both Cas9 and Cas12a can be transcribed and processed from a single array simultaneously, with the spacers for Cas9 processed by endogenous tracrRNAdependent RNase III 23 . A third architecture for multiplexed gRNA expression is to produce a single transcript encoding multiple gRNAs, each separated by RNA cleavage sequences.…”

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confidence: 99%

“…3a). Conversely, some methods use annealed oligos containing complementary overhangs that specify the order of each gRNA "unit", thus enabling their direct ligation into a digested vector 23 . Array assembly methods of this type are rapid and easy to use, and have been reported for many organisms 34,38,[42][43][44][45] .…”

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confidence: 99%

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Multiplexed CRISPR technologies for gene editing and transcriptional regulation

et al. 2020

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Multiplexed CRISPR technologies, in which numerous gRNAs or Cas enzymes are expressed at once, have facilitated powerful biological engineering applications, vastly enhancing the scope and efficiencies of genetic editing and transcriptional regulation. In this review, we discuss multiplexed CRISPR technologies and describe methods for the assembly, expression and processing of synthetic guide RNA arrays in vivo. Applications that benefit from multiplexed CRISPR technologies, including cellular recorders, genetic circuits, biosensors, combinatorial genetic perturbations, large-scale genome engineering and the rewiring of metabolic pathways, are highlighted. We also offer a glimpse of emerging challenges and emphasize experimental considerations for future studies.

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Multiplexed CRISPR technologies for gene editing and transcriptional regulation

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“…The plasmid pdCas9 54 was used for the constitutively expression of the catalytically “dead” Cas9 (dCas9), the trans-activating crRNA RNA (tracrRNA) and the CRISPR RNAs (crRNA). The double (Tb.a) and triple (Tb.a.c) spacer arrays were cloned into the Bsa I site of pdCas9 using hybridized complimentary oligonucleotides, and following the one-step scheme CRATES 68 (Supplementary Methods). Cloning procedures were performed following standard protocols of DNA digestion with restriction enzymes and ligation 62 .…”

Section: Methodsmentioning

confidence: 99%

In vivodiversification of target genomic sites using processive T7 RNA polymerase-base deaminase fusions blocked by RNA-guided dCas9

Álvarez

Mencı́a

Fernández

2019

Preprint

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32Diversification of specific DNA segments typically involve in vitro generation of large sequence 33 libraries and their introduction in cells for selection. Alternative in vivo mutagenesis systems on 34 cells often show deleterious offsite mutations and restricted capabilities. To overcome these 35 limitations, we have developed an in vivo platform to diversify specific DNA segments based on 36 protein fusions between various base deaminases (BD) and the T7 RNA polymerase (T7RNAP) 37 that recognizes a cognate promoter oriented towards the target sequence. The transcriptional 38 elongation of these fusions generates transitions C to T or A to G on both DNA strands and in 39 long DNA segments. To delimit the boundaries of the diversified DNA, the catalytically dead 40 Cas9 (dCas9) is tethered with custom-designed crRNAs as a "roadblock" for BD-T7RNAP 41 elongation. While the efficiency of this platform is demonstrated in E. coli, the system can be 42 adapted to a variety of bacterial and eukaryotic hosts. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58Directed evolution enables the selection of protein variants with improved properties as 59 therapeutics and biocatalysts 1, 2, 3 . The generation of genetic variability followed by a screening 60 process are the essential steps of directed evolution 4, 5 . In vitro mutagenesis techniques (e.g., 61error-prone PCR) can quickly produce large number of variants of the target gene but their 62 selection requires, in most cases, cloning and transformation into a host cell for expression (e.g., 63E. coli). These steps are time-consuming and labor-intensive, especially when iterative cycles 64 of mutagenesis and selection are needed. Cell-free selection methods are also feasible 6 , but 65 they are technically demanding and functional expression of complex proteins (e.g. membrane 66 proteins, multimeric enzymes) is often difficult to achieve. Hence, in vivo mutagenesis systems 67 are preferred because the generation of genetic variants, their expression and selection can 68 done in a continuous process, which accellerates directed evolution 5, 7 . 70Long-established in vivo mutagenesis methods (e.g., chemical mutagens, radiations) do not 71 target specific genes and are deleterious for the host cell due to accumulation of random 72 mutations in the genome 8 . Similarly, "mutator" host strains do not allow the concentration of the 73 mutagenic activity on the target gene, inducing the accumulation of unwanted mutations in the 74 host genome 9, 10, 11, 12 . A few in vivo mutagenesis systems with targeted specificity have been 75 reported for E. coli and yeast cells, which are the preferred hosts for cloning and expression of 76 gene libraries 5 . For example, an error-prone variant of E. coli DNA polymersase I enables the 77 mutagenesis of cloned genes in ColE1 plasmids, although it concentrates the mutations close 78 to the origin of replication 13 . A different approach involves the transformation of E. coli with 79 mutant oligonucleotide libraries, targeting one or multip...

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Genome Editing with CRISPR‐Cas Systems

Lotfy

Hsu

2022

Crispr

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Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis

Cited by 95 publications

References 58 publications

Multiplexed CRISPR technologies for gene editing and transcriptional regulation

Multiplexed CRISPR technologies for gene editing and transcriptional regulation

In vivodiversification of target genomic sites using processive T7 RNA polymerase-base deaminase fusions blocked by RNA-guided dCas9

Genome Editing with CRISPR‐Cas Systems

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