A simple and rapid method for enzymatic synthesis of CRISPR-Cas9 sgRNA libraries

Yates, Joshua D.; Russell, Robert C.; Barton, Nathaniel J; Yost, H. Joseph; Hill, John E.

doi:10.1093/nar/gkab838

Cited by 5 publications

(7 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…On the other hand, this property may have legitimate uses, such as capturing the information of the 20-bp sequence between the target recognition sequence and the cut site, bypassing the need for ligation between those two sites. For example, some protocols call for the ligation of an adapter containing an MmeI site followed by digestion and subsequent removal of the site [4,15]. Our findings indicate that this step can be avoided as MmeI can directly cut the oligo without ligation.…”

Section: Discussionmentioning

confidence: 89%

“…While developing a protocol using MmeI to generate Cas9 sgRNA libraries, we noticed an unexpected product approximately 15 bp shorter than any expected products or inputs [ 15 ]. Further analysis of the digested fragment led to the hypothesis that MmeI can cut across a double-strand break (DSB) mediated by a brief interaction between complementary 2-base overhangs on each DNA molecule.…”

Section: Resultsmentioning

confidence: 99%

“…On the other hand, this property may have legitimate uses. For example, some protocols call for the ligation of an adapter containing an MmeI site followed by digestion and subsequent removal of the site [ 4 , 15 ]. Our findings indicate that this step can be avoided as MmeI can directly cut the DNA substrate without ligation.…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

The type IIS restriction enzyme MmeI can cut across a double-strand break

et al. 2023

Self Cite

View full text Add to dashboard Cite

Background Type-IIS restriction enzymes cut outside their recognition sites, allowing them to remove their binding sites upon digestion. This feature has resulted in their wide application in molecular biology techniques, including seamless cloning methods, enzymatic CRISPR library generation, and others. We studied the ability of the Type-IIS restriction enzyme MmeI, which recognizes an asymmetric sequence TCCRAC and cuts 20 bp downstream, to cut across a double-strand break (DSB). Methods and results We used synthetic double-stranded oligos with MmeI recognition sites close to 5′ end and different overhang lengths to measure digestion after different periods of time and at different temperatures. We found that the MmeI binding and cutting sites can be situated on opposite sides of a DSB if the edges of the DNA molecules are held together by transient base-pairing interactions between compatible overhangs. Conclusion We found that MmeI can cut across a DSB, and the efficiency of the cutting depends on both overhang length and temperature.

show abstract

Section: Discussionmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

The type IIS restriction enzyme MmeI can cut across a double-strand break

et al. 2023

Self Cite

View full text Add to dashboard Cite

show abstract

“…Recent pooled or arrayed CRISPRi screening methods have had small library sizes (10 2 –10 4 ) because oligonucleotides are synthesized for targeted genes using microarrays. , However, the design and synthesis of many oligonucleotides are required based on genome information. , For random sgRNA library generation, genome-wide screening is possible at a low cost and in a relatively shorter amount of time. Our random sgRNA library with shortened TRS was prepared through PCR using two oligonucleotides with phosphorylated 5′ ends (Figure A).…”

Section: Discussionmentioning

confidence: 99%

New Target Gene Screening Using Shortened and Random sgRNA Libraries in Microbial CRISPR Interference

2023

View full text Add to dashboard Cite

CRISPR interference (CRISPRi) screening has been used for identification of target genes related to specific phenotypes using single-molecular guide RNA (sgRNA) libraries. In CRISPRi screening, the sizes of random sgRNA libraries contained with the original target recognition sequences are large (∼10 12 ). Here, we demonstrated that the length of the target recognition sequence (TRS) can be shortened in sgRNAs from the original 20 nucleotides (N 20 ) to 9 nucleotides (N 9 ) that is still sufficient for dCas9 to repress target genes in the xylose operon of Escherichia coli, regardless of binding to a promoter or open reading frame region. Based on the results, we constructed random sgRNA plasmid libraries with 5′-shortened TRS lengths, and identified xylose metabolic target genes by Sanger sequencing of sgRNA plasmids purified from Xyl − phenotypic cells. Next, the random sgRNA libraries were harnessed to screen for target genes to enhance violacein pigment production in synthetic E. coli cells. Seventeen target genes were selected by analyzing the redundancy of the TRS in sgRNA plasmids in dark purple colonies. Among them, seven genes (tyrR, pykF, cra, ptsG, pykA, sdaA, and tnaA) have been known to increase the intracellular L-tryptophan pool, the precursor of a violacein. Seventeen cells with a single deletion of each target gene exhibited a significant increase in violacein production. These results indicate that using shortened random TRS libraries for CRISPRi can be simple and cost-effective for phenotype-based target gene screening.

show abstract

“…Since then, several methods based on type II S restriction enzymes (e.g. EcoP15I or MmeI) have been developed, involving elaborate designs for the synthesis of gRNA libraries with mRNAs from chicken B cells and zebrafish heart ( 8 , 9 ). Out of these methods, only the Molecular Chipper method is designed to generate gRNA libraries with saturated coverage in particular regions ( 10 ).…”

Section: Introductionmentioning

confidence: 99%

A new method to synthesize multiple gRNA libraries and functional mapping of mammalian H3K4me3 regions

Pan

Shui

et al. 2023

Nucleic Acids Research

View full text Add to dashboard Cite

Genetic screening based on the clustered regularly interspaced palindromic repeat (CRISPR) system has been indicated to be a powerful tool for identifying regulatory genes or cis-elements. However, when applying CRISPR screens to pinpoint functional elements at particular loci, a large number of guide RNA (gRNA) spacers may be required to achieve saturated coverage. Here, we present a controlled template-dependent elongation (CTDE) method relying on reversible terminators to synthesize gRNA libraries with genomic regions of interest. By applying this approach to H3K4me3 chromatin immunoprecipitation (ChIP)-derived DNA of mammalian cells, mega-sized gRNA libraries were synthesized in a tissue-specific manner, with which we conducted screening experiments to annotate essential sites for cell proliferation. Additionally, we confirmed that an essential site within the intron of LINC00339 regulates its own mRNA and that LINC00339 is a novel regulator of the cell cycle that maintains HepG2 proliferation. The CTDE method has the potential to be automated with high efficiency at low cost, and will be widely used to identify functional elements in mammalian genomes.

show abstract

A simple and rapid method for enzymatic synthesis of CRISPR-Cas9 sgRNA libraries

Cited by 5 publications

References 34 publications

The type IIS restriction enzyme MmeI can cut across a double-strand break

The type IIS restriction enzyme MmeI can cut across a double-strand break

New Target Gene Screening Using Shortened and Random sgRNA Libraries in Microbial CRISPR Interference

A new method to synthesize multiple gRNA libraries and functional mapping of mammalian H3K4me3 regions

Contact Info

Product

Resources

About