CRISPR Genome Surgery in Stem Cells and Disease Tissues

CRISPR Genome Surgery in Stem Cells and Disease Tissues focuses uniquely on the clinical applications of CRISPR/Cas9 based technology. Topics include the latest advances in gene editing and its translational applications to various diseases, including retinal degenerative disease, recessively inherited diseases, and dominantly inherited diseases, to name a few. The book's target audience includes researchers, students, clinicians and the general public. This space that is not currently served by any existing resource, so this publication fills a gap in current literature.

• Provides a thorough review of CRISPR-Cas9, from discovery to therapy
• Covers the latest advances in gene editing and its translational applications to various diseases
• Written by global leaders in the fields of gene editing and stem cell therapy

• Language: English
• Publisher: Academic Press
• Release date: Oct 20, 2021
• ISBN: 9780128178775

Book preview

Chapter 1

The history of CRISPR: from discovery to the present

Christine L. Xu¹,²,³ and Stephen H. Tsang²,³,⁴,⁵,    ¹Department of Ophthalmology, Stanford University, Stanford, CA, United States,    ²Department of Ophthalmology, Columbia University, New York, NY, United States,    ³Jonas Children’s Vision Care, Bernard & Shirlee Brown Glaucoma Laboratory, Columbia University, New York, NY, United States,    ⁴Department of Pathology & Cell Biology, Columbia University, New York, NY, United States,    ⁵Institute of Human Nutrition, College of Physicians and Surgeons, Columbia University, New York, NY, United States

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) has revolutionized the way in which researchers edit the genome both in vivo and in vitro. CRISPR has gained worldwide attention among scientists and the public, especially after He Jiankui used CRISPR to edit the CCR5 gene in two embryos that developed into twin girls born in 2019. Oftentimes, researchers and the general public might know that CRISPR is a powerful tool for gene editing without knowing the details of its discovery and the landmark cases that led to its prominence today. This chapter will begin with an exploration of Francisco Mojica’s initial CRISPR/Cas9 discovery and end with a discussion of the current projects using this versatile gene editing tool and their implications for the future.

Keywords

CRISPR/Cas9; off-targeting; crRNA; tracrRNA; SpCas9

The beginnings of CRISPR/Cas9

The history of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 began [1] with a microbiologist by the name of Francisco Mojica, who worked at the University of Alicante in Spain [2]. His research centered around a microbe called Haloferax mediterranei, a species in the archaea family known for its tolerance to extreme salt levels. This tolerance was achieved because restriction enzymes cut the microbe’s genome in a manner that was dependent on the surrounding salt levels. In 1993, Mojica et al. discovered a unique characteristic in the genome of these H. mediterranei: repeat sequences of 30 base pairs (bp) separated by spacer sequences of roughly 36 bp. He was surprised to find that these spacers did not look like typical microbial sequences of DNA [3].

Upon further investigation, Mojica et al. discovered that these curious repeat sequences were preserved in other species as well, such as Haloferax volcanii and the more distantly related Halophilic archaea [3]. Ishino et al. reported similar findings in 1987, discovering repeat sequences in Escherichia coli [4]. Over the following 20 years, Mojica et al. would find loci for short regularly spaced repeats—which would later be renamed to CRISPR—in over 20 different microbial species [5–7]. Using his Basic Local Alignment Search Tool program, he analyzed spacers from different species and determined whether they had genetic similarity to any known DNA sequences. He found that roughly two-thirds of the 88 analyzed microbial spacers matched to viruses or conjugative plasmids related to the microbes. From this data, he hypothesized that CRISPR loci might be involved in the microbial adaptive immune system as a protective mechanism against pathogens [8].

Further investigations by Gilles Vergnaud, a geneticist who analyzed Yersinia pestis strains, led to the discovery that their tandem repeat loci were all identical, except for one distinct difference at a CRISPR locus; this particular locus had spacers matching the DNA of a prophage in the Y. pestis genome. As a result, he hypothesized that the CRISPR locus was influential in immunological defense mechanisms by retaining information of past pathogens and aggressors in their genetic memory [9].

Underlying mechanisms of CRISPR/Cas9

Subsequent CRISPR research aimed to reveal the exact manner in which this mechanism was orchestrated. Alexander Bolotin suggested that transcripts from the CRISPR locus inhibited the phage gene expression via antisense RNA inhibition [10]. This was later disproved by Barrangou et al., a group that analyzed the Streptococcus thermophilus strain and two bacteriophages [10–12]. They isolated the phage-resistant bacteria in their sample and found that the bacteria all shared the phage-derived sequences incorporated into their CRISPR locus, thereby confirming the mechanism by which CRISPR bestowed adaptive immunity to bacteria [13].

John Van der Oost devoted much of his research to the discovery of the specific molecular mechanisms involved in the CRISPR program [10]. In a 2008 study published in Science, Brouns et al. reported that five Cas proteins (collectively called Cascade) were necessary for cleaving a long precursor RNA strand derived from the CRISPR locus into a compact CRISPR RNA (crRNA) strand [14,15]. Furthermore, palindromic repeats were determined to help in the process of building the crRNA’s secondary structure. CRISPR was also programmed to target genes in the lambda phage, marking the first time in history that CRISPR was successfully programmed. The study concluded with the hypothesis that CRISPR targets DNA rather than RNA.

In 2008, Marraffini and Sontheimer provided conclusive evidence to support this hypothesis [16]. Through studying Staphylococcus epidermis, Luciano Marraffini et al. found that one of the bacterial spacers matched a nickase gene present in antibiotic-resistant Staphylococcus aureus plasmids. Plasmids normally can undergo horizontal gene transfer in bacteria and archaea via phage transduction, transformation, or conjugation. In this case, the staphylococcal conjugative plasmids could not undergo horizontal gene transfer in S. epidermis. When destroying the function of the nickase gene or the spacers in the S. epidermis CRISPR locus, however, interference no longer occurred. This suggested that CRISPR must be the causal mechanism responsible for blocking the plasmids from conjugation or plasmid transformation.

Subsequent research performed by Deveau et al. and Horvath et al. in 2008 reported more data on the mechanisms of CRISPR genome editing [17,18]. Direct observations of CRISPR in action in the S. thermophilus strain revealed that the Cas9 nuclease performed the plasmid cutting. Specifically, the cuts were made precisely at a location three nucleotides upstream of the protospacer adjacent motif (PAM). Viral DNA was also shown to be cut at the same location. Their research ultimately showed that Cas9 within the CRISPR system was responsible for making cuts at specific locations designated by the guiding crRNA sequence.

Emmanuelle Charpentier’s research revealed an additional component in the CRISPR/Cas9 machinery that worked in conjunction with crRNA [19]. Through parallel sequencing of the RNA from Streptococcus pyogenes, Dr. Charpentier's team discovered numerous novel small RNA sequences (~24 nucleotides) with complementarity to CRISPR repeats. This small trans-activating RNA (tracrRNA) was transcribed from a section of DNA adjacent to the CRISPR locus. Deleting tracrRNA caused the CRISPR system to become completely nonoperational. Therefore, Charpentier et al. discovered that the tracrRNA and crRNA hybridization and consequent cleavage by RNaseIII into a mature final product is an important step that allows the Cas9 protein to find the designated target sequence.

Virginijus Siksnys, a chemist, and his collaborators demonstrated that transferring the CRISPR locus from S. thermophilus into E. coli essentially gave E. coli the tools for adaptive immunity [20]. Specifically, E. coli could use the CRISPR locus to perform specific interference against plasmid and bacteriophage DNA, giving it protection from invaders. Furthermore, investigators discovered that the RuvC and HNH nuclease domains were responsible for making the double-stranded cuts in DNA [21,22].

Later on, Siksnys and collaborators researched CRISPR/Cas9 in a test tube [23]. In these studies, they purified the Cas9 and crRNA from S. thermophilus by tagging Cas9 with streptavidin in vivo and found that the Cas9 was able to cleave DNA in vitro in the same way that it functioned in vivo (making a double-stranded cut three nucleotides away from the PAM sequence). Furthermore, the researchers were able to use CRISPR to cleave a designated target site, specified by a customized spacer in the CRISPR locus. Mutating the two nuclease domains in Cas9 also demonstrated that the HNH domain is responsible for cleaving the strand complementary to the crRNA and that the RuvC domain is responsible for cleaving the opposite strand.

In 2012, Jinek et al. reported that a single guide RNA (sgRNA)—in other words, an engineered single RNA chimera—was able to carry out the same function as the tracrRNA:crRNA complex [24]. This breakthrough would change the future of CRISPR by making it more efficient and programmable for research purposes. Jinek et al.’s keen observation of this system’s great potential to exploit the [CRISPR] system for RNA-programmable genome editing foreshadows the future of genome engineering.

Using CRISPR/Cas9 for mammalian gene editing

In 2013, multiple labs demonstrated successful CRISPR gene editing in mammalian cells. Cong et al. published a paper in Science reporting their design of tracrRNA and pre-crRNA, used to target the human EMX1 locus in mammalian cells together with S. pyogenes Cas9 (SpCas9) [25]. This team of researchers also developed a crRNA:tracrRNA duplex with chimeric RNA, and targeted designated genomic locations in human and mouse cells (human PVALB and mouse Th). George Church’s laboratory explored CRISPR’s utility in both nonhomologous end joining (NHEJ) and homologous recombination in mammalian cells [26]. Jennifer Doudna’s laboratory published results of CRISPR gene editing in human cells using sgRNA [27]. Keith Joung’s laboratory reported targeting zebrafish embryos in vivo, using CRISPR to introduce deletions into the germline.

Future directions

It is difficult to imagine that the bacterial protective mechanism against pathogens—with its spacer sequences and curious palindromic repeats encoded into the genome—was discovered by Mojica in 1993, less than 30 years prior to the publication of this book [2]. With the large amount of research devoted to CRISPR/Cas9 experiments today, it is even harder to believe that the first CRISPR/Cas9 application in mammalian gene editing occurred only 8 years prior to this book.

Before translating the CRISPR/Cas9 genome editing tool from animal studies into clinical trials, its safety and efficacy must first be established. One of the main concerns regarding CRISPR/Cas9 technology is off-target mutagenesis, or mutations caused in locations other than the designated target locus [28]. The sgRNA used to guide Cas9 to the target site is usually 20 nucleotides long, and off-target effects could occur with even as many as 3–5 bp mismatches in the PAM distal part of the sgRNA [25,29,30]. As a result, many tools are being developed to minimize off-target effects without sacrificing CRISPR editing efficiency. Cas9 from Streptococcus aureus has less restrictions on PAM specificity, and also demonstrated high efficacy and specificity in human cells [31,32]. Campylobacter jejuni Cas9 (CjCas9) is a small Cas9 orthologue only 2.95 kb long, significantly smaller than SpCas9 (4.2 kb) [32,33]. Besides the benefits of this small size, which allows CjCas9 to fit comfortably in adeno-associated viruses (which have carrying capacities of 4.2 kb), they also have been shown to have high specificity and editing efficiency. Of equal interest is Cas12a from Lachnospiraceae bacterium ND2006 (LbCpf1) and Acidaminococcus sp. BV3L6 (AsCpf1) due to their high targeting specificity and optimal NHEJ-directed repair, achieved from creating staggered cuts with sticky ends rather than blunt double end cuts like SpCas9