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.

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Enhanced Cas12a multi-gene regulation using a CRISPR array separator

10.1101/2021.01.27.428408 ◽

2021 ◽

Author(s):

Jens P. Magnusson ◽

Antonio R. Rios ◽

Lingling Wu ◽

Lei S. Qi

Keyword(s):

Mammalian Cells ◽

Gene Editing ◽

Gc Content ◽

Gene Control ◽

Naturally Occurring ◽

Crispr Array ◽

Endogenous Genes ◽

Simultaneous Activation ◽

Type V ◽

Array Performance

AbstractThe type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple crRNAs. Here, we report that Cas12a array performance is hypersensitive to the GC content of crRNA spacers, as high-GC spacers can impair activity of the downstream crRNA. We analyzed naturally occurring CRISPR arrays and observed that repeats always contain an AT-rich fragment that separates crRNAs; we term this fragment a CRISPR separator. Inspired by this observation, we designed short, AT-rich synthetic separators (synSeparators) that successfully removed the disruptive effects between crRNAs. We demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously unknown feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.

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A Novel Type V CRISPR System with Potential for Genome Editing in the Liver

Blood ◽

10.1182/blood-2021-154505 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1862-1862

Author(s):

Gregory J. Cost ◽

Morayma Temoche-Diaz ◽

Janet Mei ◽

Cristina N. Butterfield ◽

Christopher T. Brown ◽

...

Keyword(s):

Amino Acid ◽

Genome Editing ◽

Mammalian Cells ◽

Conflicts Of Interest ◽

Attractive Alternative ◽

Vitro Assay ◽

Crispr System ◽

Type V

Abstract RNA guided CRISPR genome editing systems can make specific changes to the genomes of mammalian cells and have the potential to treat a range of diseases including those that can be addressed by editing hepatocytes. Attempts to edit the liver in vivo have relied almost exclusively on the Cas9 nucleases derived from the bacteria S treptococcus pyogenes or Staphylococcus aureus to which humans are commonly exposed. Pre-existing immunity to both these proteins has been reported in humans which raises concerns about their in vivo application. In silico analysis of a large metagenomics database followed by testing in mammalian cells in culture identified MG29-1, a novel CRISPR system which is a member of the Type V family but exhibits only 41 % amino acid identity to Francisella tularensis Cas12a/cpf1. MG29-1 is a 1280 amino acid RNA programmable nuclease that utilizes a single guide RNA comprised of a 22 nucleotide (nt) constant region and a 20 to 25 nt spacer, recognizes the PAM KTTN (predicted frequency 1 in 16 bp) and generates staggered cuts. MG29-1 was derived from a sample taken from a hydrothermal vent and it is therefore unlikely that humans will have developed pre-existing immunity to this protein. A screen for sgRNA targeting serum albumin in the mouse liver cell line Hepa1-6 identified 6 guides that generated more than 80% INDELS. The MG29-1 system was optimized for in vivo delivery by screening chemical modifications to the guide that improve stability in mammalian cell lysates while retaining or improving editing activity. Two lead guide chemistries were evaluated in mice using MG29-1 mRNA and sgRNA packaged in lipid nanoparticles (LNP). Three days after a single IV administration on-target editing was evaluated in the liver by Sanger sequencing. The sgRNA that was the most stable in the in vitro assay generated INDELS that ranged from 20 to 25% while a sgRNA with lower in vitro stability failed to generate detectable INDELs. The short sgRNA and small protein size compared to spCas9 makes MG29-1 an attractive alternative to spCas9 for in vivo editing applications. Evaluation of the potential of MG29-1 to perform gene knockouts and gene additions via non-homologous end joining is ongoing. Disclosures No relevant conflicts of interest to declare.

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Enhanced Cas12a multi-gene regulation using a CRISPR array separator

eLife ◽

10.7554/elife.66406 ◽

2021 ◽

Vol 10 ◽

Author(s):

Jens P Magnusson ◽

Antonio Ray Rios ◽

Lingling Wu ◽

Lei S Qi

Keyword(s):

Mammalian Cells ◽

Gene Editing ◽

Gc Content ◽

Guide Rna ◽

Naturally Occurring ◽

Crispr Array ◽

Endogenous Genes ◽

Simultaneous Activation ◽

Type V ◽

Array Performance

The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple guide RNA (gRNAs). Here, we report that Cas12a array performance is hypersensitive to the GC content of gRNA spacers, as high-GC spacers can impair activity of the downstream gRNA. We analyze naturally occurring CRISPR arrays and observe that natural repeats always contain an AT-rich fragment that separates gRNAs, which we term a CRISPR separator. Inspired by this observation, we design short, AT-rich synthetic separators (synSeparators) that successfully remove the disruptive effects between gRNAs. We further demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously underexplored feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.

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Gene editing in dermatology: Harnessing CRISPR for the treatment of cutaneous disease

F1000Research ◽

10.12688/f1000research.23185.1 ◽

2020 ◽

Vol 9 ◽

pp. 281

Author(s):

Catherine Baker ◽

Matthew S. Hayden

Keyword(s):

Human Genome ◽

Genome Editing ◽

Gene Editing ◽

Human Diseases ◽

Therapeutic Application ◽

Cutaneous Disease ◽

Crispr System ◽

Novel Approaches

The discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system has revolutionized gene editing research. Through the repurposing of programmable RNA-guided CRISPR-associated (Cas) nucleases, CRISPR-based genome editing systems allow for the precise modification of specific sites in the human genome and inspire novel approaches for the study and treatment of inherited and acquired human diseases. Here, we review how CRISPR technologies have stimulated key advances in dermatologic research. We discuss the role of CRISPR in genome editing for cutaneous disease and highlight studies on the use of CRISPR-Cas technologies for genodermatoses, cutaneous viruses and bacteria, and melanoma. Additionally, we examine key limitations of current CRISPR technologies, including the challenges these limitations pose for the widespread therapeutic application of CRISPR-based therapeutics.

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PAM recognition by miniature CRISPR nucleases triggers programmable double-stranded DNA target cleavage

10.1101/654897 ◽

2019 ◽

Cited By ~ 7

Author(s):

Tautvydas Karvelis ◽

Greta Bigelyte ◽

Joshua K. Young ◽

Zhenglin Hou ◽

Rimante Zedaveinyte ◽

...

Keyword(s):

Genome Editing ◽

Dependent Manner ◽

Short Sequence ◽

E Coli ◽

Defense Systems ◽

Double Stranded Dna ◽

Protospacer Adjacent Motif ◽

Type V ◽

Programmable Nucleases ◽

Cas Proteins

ABSTRACTIn recent years, CRISPR-associated (Cas) nucleases have revolutionized the genome editing field. Being guided by an RNA to cleave double-stranded (ds) DNA targets near a short sequence termed a protospacer adjacent motif (PAM), Cas9 and Cas12 offer unprecedented flexibility, however, more compact versions would simplify delivery and extend application. Here, we present a collection of 10 exceptionally compact (422-603 amino acids) CRISPR-Cas nucleases that recognize and cleave dsDNA in PAM dependent manner. Categorized as class 2 type V-F they come from the Cas14 family and distantly related type V-U3 Cas proteins found in bacteria. Using biochemical methods, we demonstrate that a 5’ T- or C-rich PAM sequence triggers double stranded (ds) DNA target cleavage. Based on this discovery, we evaluated whether they can protect against invading dsDNA in E. coli and find that some but not all can. Altogether, our findings show that miniature Cas nucleases are functional CRISPR-Cas defense systems and have the potential to be harnessed as programmable nucleases for genome editing.

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Efficient in vivo gene editing using ribonucleoproteins in skin stem cells of recessive dystrophic epidermolysis bullosa mouse model

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614775114 ◽

2017 ◽

Vol 114 (7) ◽

pp. 1660-1665 ◽

Cited By ~ 48

Author(s):

Wenbo Wu ◽

Zhiwei Lu ◽

Fei Li ◽

Wenjie Wang ◽

Nannan Qian ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Genome Editing ◽

Epidermolysis Bullosa ◽

Mammalian Cells ◽

Gene Editing ◽

Genetic Diseases ◽

Dystrophic Epidermolysis Bullosa ◽

Recessive Dystrophic Epidermolysis Bullosa

The prokaryotic CRISPR/Cas9 system has recently emerged as a powerful tool for genome editing in mammalian cells with the potential to bring curative therapies to patients with genetic diseases. However, efficient in vivo delivery of this genome editing machinery and indeed the very feasibility of using these techniques in vivo remain challenging for most tissue types. Here, we show that nonreplicable Cas9/sgRNA ribonucleoproteins can be used to correct genetic defects in skin stem cells of postnatal recessive dystrophic epidermolysis bullosa (RDEB) mice. We developed a method to locally deliver Cas9/sgRNA ribonucleoproteins into the skin of postnatal mice. This method results in rapid gene editing in epidermal stem cells. Using this method, we show that Cas9/sgRNA ribonucleoproteins efficiently excise exon80, which covers the point mutation in our RDEB mouse model, and thus restores the correct localization of the collagen VII protein in vivo. The skin blistering phenotype is also significantly ameliorated after treatment. This study provides an in vivo gene correction strategy using ribonucleoproteins as curative treatment for genetic diseases in skin and potentially in other somatic tissues.

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Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases

Nucleic Acids Research ◽

10.1093/nar/gkaa110 ◽

2020 ◽

Vol 48 (7) ◽

pp. 3722-3733 ◽

Cited By ~ 10

Author(s):

Eszter Tóth ◽

Éva Varga ◽

Péter István Kulcsár ◽

Virág Kocsis-Jutka ◽

Sarah Laura Krausz ◽

...

Keyword(s):

Genome Editing ◽

Mammalian Cells ◽

Genome Engineering ◽

Plant Cells ◽

Dependent Manner ◽

Protospacer Adjacent Motif

Abstract The widespread use of Cas12a (formerly Cpf1) nucleases for genome engineering is limited by their requirement for a rather long TTTV protospacer adjacent motif (PAM) sequence. Here we have aimed to loosen these PAM constraints and have generated new PAM mutant variants of the four Cas12a orthologs that are active in mammalian and plant cells, by combining the mutations of their corresponding RR and RVR variants with altered PAM specificities. LbCas12a-RVRR showing the highest activity was selected for an in-depth characterization of its PAM preferences in mammalian cells, using a plasmid-based assay. The consensus PAM sequence of LbCas12a-RVRR resembles a TNTN motif, but also includes TACV, TTCV CTCV and CCCV. The D156R mutation in improved LbCas12a (impLbCas12a) was found to further increase the activity of that variant in a PAM-dependent manner. Due to the overlapping but still different PAM preferences of impLbCas12a and the recently reported enAsCas12a variant, they complement each other to provide increased efficiency for genome editing and transcriptome modulating applications.

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