CRISPR Gene Editing: Cas9 and Beyond

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CRISPR, an acronym for clustered regularly interspaced short palindromic repeats, is a family of genes that first evolved in prokaryotic organisms such as bacteria and archaea to defend against infectious phages.¹ Analogous to eukaryotic adaptive immune memory, CRISPR sequences derive from bacteriophages that previously infected prokaryotes; bacteria use their CRISPR sequences and nucleases called CRISPR associated (Cas) proteins to detect and destroy familiar bacteriophages.¹ Today, researchers build on the mechanisms of prokaryotic CRISPR systems to engineer CRISPR-Cas mediated gene editing technologies, which use Cas proteins and guide RNAs to block, cut, or edit target genes.¹

How Gene Editing Works

Originating from the bacterium Streptococcus pyogenes, Cas9 was the first Cas protein that scientists repurposed for gene editing.¹ CRISPR-Cas9 technology uses a single guide RNA (sgRNA) to target and cleave DNA. Researchers engineer target-specific sgRNAs by combining two RNA molecules from the bacterial CRISPR system: a sequence that recognizes a specific location in the DNA (crRNA) and a sequence that acts as a binding scaffold for Cas9 (tracrRNA).¹ The modifiable sgRNA sequence allows scientists to program a CRISPR-Cas9 system to target any DNA sequence of interest if it is near a Cas-specific DNA sequence called a protospacer-adjacent motif (PAM).^1,2 The PAM initiates Cas9-DNA binding, the sgRNA invades the double helix and hybridizes with the target DNA, and Cas9 breaks the unwound double-stranded target DNA immediately in front of the PAM. Repair mechanisms, namely homology-directed repair (HDR) and nonhomologous end joining (NHEJ), repair the break, which can alter the target gene’s biological function.³ 

For instance, researchers can use the different repair mechanisms to their advantage to intentionally insert a desired sequence change via template-dependent HDR or introduce random changes through template-independent NHEJ. If scientists provide the cellular repair machinery with a target gene template that contains a mutation, such as a disease-relevant mutation, the HDR process will incorporate the templated mutation into the genome after Cas9 cleavage.^1,2 In contrast, the more error prone NHEJ pathway repairs the Cas9 cut without a template by introducing random insertions and deletions (indels) that ultimately disrupt gene expression or lead to loss of function.^1,2 Either of these approaches enable researchers to investigate how silencing or editing a targeted gene affects its downstream pathways.¹

         

Overcoming Limitations of CRISPR

Researchers often struggle with Cas9 cutting the DNA in off-target locations, which can lead to translocations, unexpected large deletions, and unwanted gene expression changes.⁴ To avoid such off-target side effects, researchers engineer alternatives to Cas9.⁵

Table 1. Overview of Cas9 and common Cas9 alternatives^5-7

Cas12a

Cas12a offers multiple advantages over Cas9 (Table 1).⁸ It produces staggered rather than blunt DNA breaks,⁹ which favor HDR mechanisms instead of both NHEJ and HDR.⁵ Additionally, Cas12a does not need a tracrRNA, and possesses RNase activity that can process a single pre-crRNA array containing several guide RNAs for easier multigene editing than Cas9.¹⁰ Cas12a also has reduced off-target effects compared to Cas9,¹¹ and is uniquely capable of PAM-independent single-stranded DNA degradation, which is useful for diagnostic applications.^5,12 

Cas13

Cas13 differs from Cas9 and Cas12a as it cleaves single-stranded RNA rather than DNA. Working with its guide sequence, it targets RNA in eukaryotic cells for degradation with high efficiency. This is a promising approach for transcriptome engineering, as it modulates gene expression without altering the DNA sequence.¹³ 

Genome Modifications with Base or Prime Editing

Base editors and prime editors are new generation CRISPR-based technologies that rely on catalytically impaired Cas proteins called nickases, which researchers fuse to other enzymes that modify DNA.² Because Cas nickases are less active than the traditional Cas nucleases, they introduce single-stranded DNA breaks (or nicks) instead of double-stranded breaks. These nicks do not typically recruit DNA repair machinery, which allows fusion enzymes to act on the DNA with greater editing accuracy than repair-mediated methods.²

What is base editing?

Base editing uses modified versions of DNA cutting Cas proteins that retain their DNA binding abilities without introducing double-stranded breaks.¹⁴ These catalytically impaired Cas proteins guide an adenine or cytidine deaminase enzyme to a specific site in the DNA, where they cause C G to T A or A T to G C conversions.¹⁵ In theory, base editing can correct any transition mutation (A to G, or C to T),¹⁶ which represent roughly 30 percent of known disease alleles and are the single largest class of human disease causing mutations.¹⁴

What is prime editing?

For prime editing, a nuclease-impaired Cas protein recruits a reverse transcriptase to the desired DNA sequence without introducing double-stranded breaks.¹⁷ A reverse transcriptase converts RNA into DNA. In the context of prime editing, the reverse transcriptase replaces or inserts a DNA sequence encoded by a prime editor guide RNA (pegRNA).¹⁷ These highly versatile prime editors are not limited to the specific pairing of base editors and can catalyze the switch between any base. They can also generate insertions up to 44 base pairs (bps) and deletions of up to 80 bps.¹⁸ 

Transcriptome Editing with CRISPR Modulation and dCas

CRISPR modulation changes gene expression without introducing changes to the DNA sequence.¹⁹ Scientist fuse a catalytically inactive Cas nuclease (dead Cas or dCas) with either a repressor domain for CRISPR interference (CRISPRi) or with a transcriptional activation domain for CRISPR activation (CRISPRa).¹⁹ The dCas fusion protein binds to the DNA target sequence without introducing double-stranded breaks, recruits small regulatory RNAs to the locus, and causes either gene expression knockdown (CRISPRi via the repressor domain) or gene expression activation (CRISPRa).²⁰ 

New CRISPR Technology Applications

Base editing has powerful medical potential because of its low risk of creating insertions and deletions. It has been paired with CAR-T cell therapy in clinical trials to treat otherwise untreatable leukemia cases.²¹ 

Because prime editing can correct most mutations, excluding large duplications, deletions, insertions, or chromosomal rearrangements, researchers are investigating its ability to treat a variety of disorders, ranging from inherited diseases to cancer.²² 

Finally, preclinical CRISPRi and CRISPRa applications allow researchers to perform large screens of the genetic networks underlying diseases with unknown etiologies, such as neurodegenerative disorders.^23,24