Are you sure of checking for the PAM sequence? Any GG on any strand (+ or -) could be used as a PAM sequence. So is highly probable to find a GG.

If you didn't get any GG sequence, you can use other PAM sequence (upstream or downtream) adjacent to your gene and make the Cas9 cut there. But you will need a donor DNA with homology so it can recombine.

Play with the lenght of the homologies to make editions.

Article Metabolic engineering of Escherichia coli using CRISPR-Cas9 ...

thx for the answer!

There are a lot of different Cas9 types, with each a different PAM motif that is recognized. The NGG is for the Cas9 protein of S.pyogenes, but there are a lot of different bacteria with Cas9 protein, and so differents PAM (N.meningitidis, S.aureus,....)

And recently, the CpfI protein, from different bacteria, which work like Cas9, and has different PAM motif...

So you should be able, with all these possibilities, to find the right Cas9 protein for your specific sequence.

Maybe you can use this software that we built to find, in your genomic sequence, a PAM motif whith different Cas9...

http://crispor.tefor.net/crispor.py

Dear Esteemed Colleague,

Thank you for your query regarding the Protospacer Adjacent Motif (PAM) sequence requirement in CRISPR-Cas9 genome editing and how researchers navigate the challenge of targeting genes without an adjacent PAM sequence. The CRISPR-Cas9 system, a revolutionary tool for precise genome editing, indeed requires a PAM sequence near the target site for the Cas9 nuclease to bind and introduce double-stranded breaks. The presence of a PAM sequence is a critical determinant of the target site's eligibility for CRISPR-Cas9 editing. Let's explore how scientists address the limitations posed by PAM sequence requirements:

1. Cas9 Variants with Different PAM Requirements

The most common strategy to overcome PAM limitations involves using Cas9 variants or orthologs from different bacterial species, each with unique PAM requirements. For instance, while the commonly used Streptococcus pyogenes Cas9 (SpCas9) recognizes the PAM sequence 5'-NGG-3', other variants such as Staphylococcus aureus Cas9 (SaCas9) prefer 5'-NNGRRT-3', and Streptococcus thermophilus Cas9 (StCas9) recognizes 5'-NNAGAAW-3'. These variations in PAM preferences increase the range of potential target sites across the genome.

2. Engineered Cas9 Variants

Scientists have engineered Cas9 proteins with altered PAM specificities to expand the targeting range further. Through protein engineering, variants of Cas9 have been developed to recognize alternative PAM sequences not recognized by the wild-type Cas9. This approach has significantly broadened the applicability of CRISPR-Cas9 technology, allowing for targeting regions previously inaccessible due to the absence of suitable PAM sequences.

3. Utilizing Cpf1 (Cas12a)

Beyond Cas9, researchers have explored other CRISPR-associated nucleases like Cpf1 (now referred to as Cas12a), which recognizes different PAM sequences (e.g., 5'-TTTN-3') and offers an alternative for targeting sites without the typical Cas9 PAM sequences. Cas12a not only broadens the targeting range but also generates staggered cuts, providing additional advantages in certain genome editing applications.

4. Base Editing and Prime Editing

Base editing, a technique derived from CRISPR-Cas9 technology, enables the direct conversion of one DNA base pair into another without introducing double-stranded breaks, thereby circumventing the need for a PAM sequence in close proximity to the target site. Prime editing, a more recent innovation, extends this capability further, allowing for precise editing without strict PAM sequence requirements, offering an elegant solution to the PAM constraint.

5. Synthetic PAM Creation

In some instances, researchers have employed strategies to introduce a synthetic PAM sequence into the target genome through homologous recombination or other means, thereby creating a suitable site for Cas9 binding and activity. Although more complex, this approach can be viable for certain applications where other methods are not suitable.

In conclusion, while the requirement for a PAM sequence presents a challenge in CRISPR-Cas9 genome editing, the scientific community has developed several innovative strategies to overcome this limitation. Through the use of Cas9 variants, engineered nucleases, alternative CRISPR systems, and novel genome editing techniques, researchers can now target a broader range of genomic sequences than ever before.

I trust this explanation provides a clear understanding of how researchers navigate the challenge of PAM sequence requirements in CRISPR-Cas9 genome editing and continues to advance the field of genetic engineering.

Best regards.

l Take a look at this protocol list; it could assist in understanding and solving the problem.