![]() The CRISPR-Cas13a system has been utilized for various applications including messenger RNA (mRNA) knockdown, mRNA live imaging, and RNA base editing ( Abudayyeh et al., 2017, 2019 Cox et al., 2017). Cas13a from Leptotrichia wadei is a well-characterized member, which uses a single crRNA containing an ~36-nucleotide direct repeat (DR) and a 28- to 30-nucleotide spacer to target the spacer complementary region within single-stranded RNA (ssRNA) substrates ( Abudayyeh et al., 2016, 2017 East-Seletsky et al., 2016). CRISPR-Cas9 and its derivatives have been extensively developed for therapeutic applications ( Doudna, 2020 Fellmann et al., 2017 Jinek et al., 2012 Knott and Doudna, 2018 Ran et al., 2015 Shalem et al., 2014 Zhou et al., 2014).īesides targeting DNAs, there are also CRISPR-Cas systems targeting RNA, such as the Cas13 family, including Cas13a (previously known as C2c2), Cas13b, Cas13c, and Cas13d ( Abudayyeh et al., 2017 Cox et al., 2017 East-Seletsky et al., 2017 Knott et al., 2017). ![]() HDR requires exogenous DNA templates that can direct the replacement of the native DNA sequence with the designed one. ![]() NHEJ often leads to nucleotide insertions or deletions (indels) around the DSB site ( Jinek et al., 2012), disrupting the open reading frame of the target gene. The resulting ribonucleoprotein (RNP) targets the DNA substrate containing the complementary sequence, and Cas9 then cleaves the DNA, generating site-specific double-strand breaks (DSBs) that can be repaired through non-homologous end-joining (NHEJ) or homology-directed repair (HDR) in cells. Cas9 can bind to single-guide RNA (sgRNA) containing an ~20-nucleotide sequence designed to match the targeted genes ( Cong et al., 2013 Jiang and Doudna, 2017 Jinek et al., 2012 Mali et al., 2013). Among the diverse CRISPR-Cas systems, CRISPR-Cas9 from Streptococcus pyogenes is the most popular ( Koonin et al., 2017). CRISPR-Cas systems, an adaptive immune system widespread in bacteria and archaea ( Barrangou et al., 2007), are comprised of Cas proteins and CRISPR RNA (crRNA), which together target and cleave invading genetic materials through RNA-guided endonuclease activity. Developing effective delivery methods that can mediate entry of membrane-impermeable macromolecules into cells is essential to unlock the therapeutic potential of targeting intracellular substrates.ĬRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are a major class of potential therapeutic tools. These findings establish modular delivery systems based on single-chain bacterial toxins for delivery of membrane-impermeable therapeutics into targeted cells.ĭevelopment of protein-based biological therapeutics has revolutionized treatment for many human diseases, yet since the cell membrane is a formidable barrier, these therapeutics are largely limited to cell surface targets. ![]() Delivery of Cas9, together with guide RNA expression, generates mutations at the targeted genomic sites in cell lines and induced pluripotent stem cell (iPSC)-derived human neurons. Delivery of Cre recombinase modifies the reporter loci in cells. Delivery of Cas13a and CasRx, together with guide RNA expression, reduces mRNAs encoding GFP, SARS-CoV-2 fragments, and endogenous proteins PPIB, KRAS, and CXCR4 in multiple cell lines. The system can deliver large protein cargoes including Cas13a, CasRx, Cas9, and Cre recombinase into cells in a receptor-dependent manner, although delivery of ribonucleoproteins containing guide RNAs is not successful. Here, we develop protein-based delivery systems utilizing detoxified single-chain bacterial toxins such as diphtheria toxin (DT) and botulinum neurotoxin (BoNT)-like toxin, BoNT/X, as carriers. Targeted delivery of therapeutic proteins toward specific cells and across cell membranes remains major challenges.
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